Dual Inhibition of MDM2 and XPO1 Synergizes to Induce Apoptosis in Acute Myeloid Leukemia Progenitor Cells with Wild-Type TP53 through Nuclear Accumulation of p53 and Suppression of c-Myc

Background. MDM2 is frequently overexpressed in acute myeloid leukemias (AML) and suppresses p53-mediated apoptosis while p53 mutations are relatively rare in AML. MDM2 inhibitors as a monotherapy have shown limited efficacy in clinical trials in AML (~25% response rate) (Andreeff, Clin Cancer Res 2015). XPO1 transports around 300 proteins, including p53 and other tumor suppressors, from the nucleus to the cytoplasm. Overexpression of XPO1 is associated with unfavorable outcomes in AML (Kojima, Blood 2013). p53 activation or XPO1 inhibition have been reported to decrease c-Myc protein levels through diverse mechanisms (Porter Mol Cell 2017 and Tabe PLoSOne 2015). Objective: We investigated anti-leukemia effect of dual MDM2 and XPO1 inhibition, with the intent to maximize the pro-apoptotic functions of p53, using the MDM2 inhibitor milademetan (Daiichi-Sankyo), and selinexor, a recently FDA-approved XPO1 inhibitor or its analog eltanexor (Karyopharm). Results: Treatment with milademetan and selinexor (1:1 molar ratio) induced synergistic apoptosis in AML cell lines with wild-type p53 (ED50, 89.3 ± 18.6 nM, combination index (CI), 0.60 ± 0.08). Activity in p53 mutant AML required 40-fold higher ED50 (3572 ± 1986 nM), reflected in an antagonistic CI of 6.94 ± 3.06. Knockdown of wild-type p53 by shRNA in OCI-AML3 (OCI-AML3 shp53) cells or presence of TP53 mutation (p.R248W) in MOLM-13 cells eliminated the synergistic effects, suggesting that normal p53 function is a major determinant of sensitivity to combined treatment. Next, we treated primary AML samples with milademetan and selinexor or eltanexor and observed that effects were mutation-agnostic (e.g. RAS and FLT3) except for TP53. Combined treatment significantly reduced AUC determined by absolute live cell numbers compared to each drug alone, and induced synergistic apoptosis in primary AML samples with wild-type p53 (ED50 values, 27.2 - 937.4 nM, CI, 0.51 ± 0.07), with similar efficacies in complex and non-complex karyotype AMLs (279.6 ± 94.7 vs 256.6 ± 56.4 nM, P = 0.84). In contrast, combined treatment showed antagonistic effects in primary AML samples with loss-of-function TP53 mutations (CI > 1.0). Immature CD34+CD38- AML cells were more susceptible to combined treatment than CD34- AML cells (apoptosis induction, 76.2 ± 6.7% vs 47.5 ± 6.8%, P = 0.0002) Mechanistically, combined inhibition increased p53 protein levels and accumulated p53 but not MDM2 protein in the nucleus compared to each drug alone. Combined treatment induced more TP53 target genes (MDM2, CDKN1A, BBC3, FAS and Bax) in OCI-AML3 cells with control shRNA compared with OCI-AML3 shp53 cells. Combinatorial inhibition showed much enhanced reduction of c-Myc mRNA and protein levels in OCI-AML3 shC cells compared with OCI-AML3 shp53 cells (82% vs 32%). In confirmation, combined inhibition reduced c-Myc protein levels profoundly in wild-type p53 primary AMLs (ANOVA P < 0.0001). In contrast, c-Myc reduction was not observed in primary AMLs with p53-inactivating mutations. Intriguingly, OCI-AML3 cells overexpressing c-Myc by lentiviral transduction showed greater sensitivity to XPO1 inhibitors and the combination compared to empty-vector controls, and baseline levels of c-Myc protein also negatively correlated with ED50 for combined treatment in primary AML samples (Spearman R = -0.5357, P = 0.0422). Conclusion: These preclinical data suggest that dual inhibition of MDM2 and XPO1 induces synergistic apoptosis through accumulation of nuclear p53 and suppression of c-Myc in wild-type p53 AMLs. A clinical trial testing this concept in AML is under development. Disclosures Ishizawa: Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed. Daver:Novartis: Consultancy, Research Funding; Agios: Consultancy; Jazz: Consultancy; Hanmi Pharm Co., Ltd.: Research Funding; Pfizer: Consultancy, Research Funding; Astellas: Consultancy; Immunogen: Consultancy, Research Funding; Forty-Seven: Consultancy; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Servier: Research Funding; Incyte: Consultancy, Research Funding; NOHLA: Research Funding; Glycomimetics: Research Funding; BMS: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; Celgene: Consultancy; Otsuka: Consultancy. Lesegretain:Daiichi-Sankyo Inc.: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy.

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Exportin-1 (XPO1) Inhibition Sequesters p53 from MDM2 and MDM4 and Is Highly Synergistic with MDM2 Inhibition in Inducing Apoptosis in Wild-Type p53 Acute Myeloid Leukemias

Blood ◽

10.1182/blood-2020-140768 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-24

Author(s):

Yuki Nishida ◽

Jo Ishizawa ◽

Edward Ayoub ◽

Rafael Heinz Montoya ◽

Vivian Ruvolo ◽

...

Keyword(s):

Board Of Directors ◽

Combination Treatment ◽

Research Funding ◽

Combined Treatment ◽

Wild Type ◽

Advisory Committees ◽

Ki 67 ◽

Protein Levels ◽

Dual Inhibition ◽

Mdm2 Inhibitor

The tumor suppressor p53 is inactivated in virtually all cancers, including leukemias, by mutations or deletion of the TP53 gene, or overexpression of negative regulators (e.g., MDM2, MDM4, and XPO1). MDM2 and MDM4 are frequently overexpressed in acute myeloid leukemia (AML), with the highest MDM4 expression as compared to other malignancies. XPO1 transports ~300 proteins, including p53, from the nucleus to the cytoplasm and MDM2 is also a cargo protein transported by XPO1.We previously reported high synergism by MDM2 and XPO1 inhibition in AML (Kojima et al., Blood 2013), with the underlying mechanism yet to be fully investigated. Wp53 was highly accumulated in the nucleus by combined treatment in OCI-AML3 and primary AML cells with MDM2 inhibitor milademetan (DS-3032b) and XPO1 inhibitor selinexor (KPT-330), compared to treatment with individual drugs. Upon MDM2 and XPO1 inhibition, MDM2 was exclusively localized in the cytoplasm, not in the nucleus. Intriguingly, MDM4 also localized exclusively in the cytoplasm, and the dual inhibition markedly reduced the level of cytoplasmic MDM4 (Fig.1). Data suggest that the dual inhibition of MDM2 and XPO1 maximizes the transcriptional activity of p53 by sequestering MDM2 and MDM4 in the cytoplasm, with massive p53 induction in the nucleus. Indeed, the combination treatment dramatically induced p53 targets CDKN1A and MDM2 (i.e.,55-fold and 25-fold, respectively). WPathway analysis from RNA seq of OCI-AML3 cells treated with milademetan, selinexor, and the combination revealed the TP53 pathway was the top upregulated pathway compared with control, or single agent treatments. E2F targets, G2M checkpoint genes and MYC targets were the principal downregulated pathways by the combination treatment. Cell cycle analysis measuring EdU/DNA, Ki-67, p53, p21, and active caspase-3 revealed elimination of S-phase and a population with the highest Ki-67 levels at G2/M phase, with increased percentages of cells in G0 and G2/M phases. The combination treatment markedly reduced Ki-67 levels, suggesting the disruption of DNA synthesis and cell cycle arrest. Furthermore, p53 and p21 levels were increased in both G0 and G2/M cells, along with increased active caspase-3 levels, suggesting apoptosis induction in both highly proliferating and quiescent AML cells. WNext to validate c-Myc inhibition by the combination treatment, we used OCI-AML3 cells transduced with shRNA control (ShC) and shRNA for p53 (Shp53), and MOLM-13 cells with wild-type p53 (WT) and with TP53 p.R248W/R213* mutation (MT), obtained through long-term exposure to MDM2 inhibitor. c-Myc protein levels were significantly reduced in OCI-AML3 ShC cells and MOLM-13 WT cells, but not in OCI-AML3 Shp53 cells or MOLM-13 MT cells. Combined treatment synergistically reduced MYC mRNA and c-Myc protein levels both in the cytoplasm and the nucleus. Consistently, the combination treatment reduced c-Myc levels in primary AML cells with wild-type TP53 as opposed to those with TP53 mutations. Overexpression of c-Myc in OCI-AML3 cells conferred increased susceptibility to the combination treatment. Finally, c-Myc protein levels at baseline had negative correlation with the respective ED50 concentrations that induced apoptosis in 50% of AML blasts. In conclusion: we identified strikingly increased transcriptional activity of p53 which was retained in the nucleus and sequestration of MDM2 in the cytoplasm as novel mechanisms of combined MDM2/XPO1 inhibition. In addition, high c-Myc baseline levels were associated with response to the combinatorial treatment, which also markedly reduced nuclear and cytoplasmic c-Myc levels due to MYC repression by p53 activation. These findings support the translation of combined MDM2 and XPO1 inhibition into clinical trials. Disclosures Daver: Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lesegretain:Daiichi-Sankyo Inc.: Current Employment. Shacham:Karyopharm: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: (8999996, 9079865, 9714226, PCT/US12/048319, and I574957) on hydrazide containing nuclear transport modulators and uses, and pending patents PCT/US12/048319, 499/2012, PI20102724, and 2012000928) . Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

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TP73 As Novel Determinant of Resistance to BCL-2 Inhibition in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-127182 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1251-1251

Author(s):

Yuki Nishida ◽

Jo Ishizawa ◽

Vivian Ruvolo ◽

Feng Wang ◽

Koichi Takahashi ◽

...

Keyword(s):

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Mrna Levels ◽

Wild Type ◽

Advisory Committees ◽

Protein Levels ◽

Enhanced Sensitivity ◽

Equity Ownership ◽

Acute Myeloid

Background. BCL-2 inhibition is a novel and highly effective treatment modality in acute myeloid leukemias (AML). AML patients with IDH1/2 mutations are highly sensitive to BCL-2 inhibition by venetoclax (VEN) (Chen et al Nat Med 2015). High expression levels of the BCL-2 family proteins MCL-1 or BCL-XL, or knockout of TP53 have been reported to confer resistance to BCL-2 inhibition (Pan et al. Cancer Cell 2017, Nechiporuk et al. Cancer Discov 2019). p73 is one of the p53 family transcription factors and generates two isoforms, transactivation p73 (TAp73) and the N-terminally truncated ΔNp73. TAp73 shares a homologous N-terminal activation domain with p53 and has pro-apoptotic function similar to p53. ΔNp73 lacks an activation domain and has a dominant negative effect on the DNA binding of TAp73 and more importantly, of p53.TP73 is expressed in AML except in acute promyelocytic leukemias. However, the associations of TP73 isoforms with clinical and genetic characteristics or sensitivity to BCL-2 inhibition in AML have not been explored. Results. We determined copy numbers of TAp73 and ΔNp73 mRNA levels in AML samples (N = 78) and normal CD34+ hematopoietic cells (HPC) using droplet digital PCR and investigated their clinical and biological relevance. Both TP73 isoforms were expressed in AML, with TAp73 expression being 50-fold higher in AML than in CD34+ HPC (P = 0.027); no difference seen for ΔNp73 (P = 0.80), suggesting that TAp73 is aberrantly expressed in AML cells. ΔNp73 and TAp73 mRNA levels were highly correlated (R2 = 0.72, P < 0.0001). AML samples had 10-fold more abundant TAp73 than ΔNp73 mRNA levels (P = 0.0017) and isoforms were not associated with disease status (de novo vs relapsed/refractory) or cytogenetic groups, and were mutation-agnostic, except for IDH1/2. IDH1/2 mutant AML showed lower levels of TAp73 and ΔNp73 than those with wild-type IDH1/2 (P = 0.06 and P = 0.007 for TAp73 and ΔNp73, respectively). In a separate dataset, we observed repressed TP73 in IDH1/2 mutant vs. wild-type AML samples (P = 0.073) by RNAseq analysis (N = 47). Mechanistically, treatment with cell permeable octyl-(R)-2HG, the oncometabolite of mutant IDH1/2, reduced both TAp73 and ΔNp73 and increased susceptibility to VEN. Lentiviral knockdown of p73 in OCI-AML3 cells resulted in enhanced sensitivity to VEN with no significant changes in MCL-1 and p53 protein levels, or TP53 targets (MDM2, CDKN1A, FAS and BBC3). VEN resistant AML cells (MOLM-13 and MV4;11) generated through long-term culture with VEN expressed highly elevated TP73 mRNA and protein levels without significant changes in p53 or TP53 target changes, suggesting that elevated p73 could confer resistance to VEN independent of p53 function (Figure). Knockdown of TP73 showed increased protein levels of SDHB, UQCRC2 and ATP5A, components of mitochondrial respiratory chain complex II, III and V, indicating increased dependency on oxdative phosphorylation by depleting p73. Overexpression of TAp73α by lentiviral gene transfer minimally increased VEN-induced apoptosis, while ΔNp73γ overexpression conferred striking resistance to VEN in MOLM-13 cells, suggesting p73 isoform-specific dependency of VEN sensitivity/resistance. The combination of 5'-azacitidine (5'-aza) and VEN decreased ΔNp73 level by 50%. Conclusion. Repression of TP73 in IDH1/2 mutant AML, and downregulation of TP73 by the oncometabolite 2-HG were associated with enhanced sensitivity to VEN, suggesting that TP73 determines AML susceptibility to BCL-2 inhibition. VEN resistant cells massively overexpressed TP73, and TP73 knockdown restored sensitivity to VEN. Specifically, overexpression of the ΔNp73γ isoform resulted in induced VEN resistance. ΔNp73 levels were also reduced by combining VEN with 5'-aza. Results may explain the high sensitivity of IDH1/2 mutant AML to VEN as consequence of downregulation of TP73 by 2-HG, and establish the mechanism of synergistic effect by VEN + 5'-aza combination and overexpression of p73 as a novel resistance mechanism to BCL-2 inhibition. Disclosures Ishizawa: Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed. Takahashi:Symbio Pharmaceuticals: Consultancy. Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Oncolyze: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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p53 Mediated Bone Marrow Mesenchymal Stem Cell Expansion Supports Acute Myeloid Leukemia Development

Blood ◽

10.1182/blood-2019-127245 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 523-523

Author(s):

Rasoul Pourebrahimabadi ◽

Zoe Alaniz ◽

Lauren B Ostermann ◽

Hung Alex Luong ◽

Rafael Heinz Montoya ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Osteoblast Differentiation ◽

Hematopoietic Cells ◽

Advisory Committees ◽

Equity Ownership ◽

Mdm2 Inhibitor ◽

Acute Myeloid

Acute myeloid leukemia (AML) is a heterogeneous disease that develops within a complex microenvironment. Reciprocal interactions between the bone marrow mesenchymal stem/stromal cells (BM-MSCs) and AML cells can promote AML progression and resistance to chemotherapy (Jacamo et al., 2014). We have recently reported that BM-MSCs derived from AML patients (n=103) highly express p53 and p21 compared to their normal counterparts (n=73 p<0.0001) (Hematologica, 2018). To assess the function of p53 in BM-MSCs, we generated traceable lineage specific mouse models targeting Mdm2 or Trp53 alleles in MSCs (Osx-Cre;mTmG;p53fl/fl and Osx-Cre;mTmG;Mdm2fl/+) or hematopoietic cells (Vav-Cre;mTmG;p53fl/fl and Vav-Cre;mTmG;Mdm2fl/+). Homozygote deletion of Mdm2 (Osx-Cre;Mdm2fl/fl) resulted in death at birth and displayed skeletal defects as well as lack of intramedullary hematopoiesis. Heterozygote deletion of Mdm2 in MSCs was dispensable for normal hematopoiesis in adult mice, however, resulted in bone marrow failure and thrombocytopenia after irradiation. Homozygote deletion of Mdm2 in hematopoietic cells (Vav-Cre;Mdm2fl/fl) was embryonically lethal but the heterozygotes were radiosensitive. We next sought to examine if p53 levels in BM-MSCs change after cellular stress imposed by AML. We generated a traceable syngeneic AML model using AML-ETO leukemia cells transplanted into Osx-Cre;mTmG mice. We found that p53 was highly induced in BM-MSCs of AML mice, further confirming our findings in primary patient samples. The population of BM-MSCs was significantly increased in bone marrow Osx-Cre;mTmG transplanted with syngeneic AML cells. Tunnel staining of bone marrow samples in this traceable syngeneic AML model showed a block in apoptosis of BM-MSCs suggesting that the expansion of BM-MSCs in AML is partly due to inhibition of apoptosis. As the leukemia progressed the number of Td-Tomato positive cells which represents hematopoietic lineage and endothelial cells were significantly decreased indicating failure of normal hematopoiesis induced by leukemia. SA-β-gal activity was significantly induced in osteoblasts derived from leukemia mice in comparison to normal mice further supporting our observation in human leukemia samples that AML induces senescence of BM-MSCs. To examine the effect of p53 on the senescence associated secretory profile (SASP) of BM-MSCs, we measured fifteen SASP cytokines by qPCR and found significant decrease in Ccl4, Cxcl12, S100a8, Il6 and Il1b upon p53 deletion in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) compared to p53 wildtype mice. To functionally evaluate the effects of p53 in BM-MSCs on AML, we deleted p53 in BM-MSCs (Osx-Cre;mTmG;p53fl/fl) and transplanted them with syngeneic AML-ETO-Turquoise AML cells. Deletion of p53 in BM-MSCs strongly inhibited the expansion of BM-MSCs in AML and resulted in osteoblast differentiation. This suggests that expansion of BM-MSCs in AML is dependent on p53 and that deletion of p53 results in osteoblast differentiation of BM-MSCs. Importantly, deletion of p53 in BM-MSCs significantly increased the survival of AML mice. We further evaluated the effect of a Mdm2 inhibitor, DS-5272, on BM-MSCs in our traceable mouse models. DS-5272 treatment of Osx-cre;Mdm2fl/+ mice resulted in complete loss of normal hematopoietic cells indicating a non-cell autonomous regulation of apoptosis of hematopoietic cells mediated by p53 in BM-MSCs. Loss of p53 in BM-MSCs (Osx-Cre;p53fl/fl) completely rescued hematopoietic failure following Mdm2 inhibitor treatment. In conclusion, we identified p53 activation as a novel mechanism by which BM-MSCs regulate proliferation and apoptosis of hematopoietic cells. This knowledge highlights a new mechanism of hematopoietic failure after AML therapy and informs new therapeutic strategies to eliminate AML. Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Bueso-Ramos:Incyte: Consultancy. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; NIH/NCI: Research Funding; CPRIT: Research Funding; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy. OffLabel Disclosure: Mdm2 inhibitor-DS 5272

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Targeting Misfolded p53 and p53 Aggregation to Overcome Resistance to Apoptosis in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-130900 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3786-3786

Author(s):

Michael Andreeff ◽

Jianfang Zeng ◽

Alice Soragni ◽

Vivian Ruvolo ◽

Bing Z Carter ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Protein Aggregation ◽

Board Of Directors ◽

Protein Misfolding ◽

Myeloid Leukemia ◽

Research Funding ◽

Advisory Committees ◽

Equity Ownership ◽

Mdm2 Inhibitor ◽

Acute Myeloid

The function of wild-type (wt) p53 in acute myeloid Leukemia (AML) is suppressed by MDM2, MDM4 and XPO-1 (Andreeff et al, Exp Hematol, 2016). We propose that wt p53 protein misfolding and cytosolic localization are contributing to its inactivation in AML. Immunofluorescence staining with OpalR TSA amplification demonstrated that p53 is localized both in the nucleus and in the cytosol of AML cells with prominent para-nuclear accumulation. We show here that misfolded wt p53 is localized mainly in the cytoplasm of AML cells, similar to what we reported for mutant (mt) p53 previously (Zeng et al, Blood, 2016). p53 misfolding promotes its aggregation which was recently reported as a novel mechanism promoting loss of its anti-tumor functions (Xu et al, Nat Chem Biol, 2011; Soragni et al, Cancer Cell, 2016). A pro-aggregating segment in the p53 DNA binding domain is exposed when p53 is misfolded. We showed that ReACp53, a cell permeable peptide designed to inhibit the aggregation of this segment, induced apoptosis in ovarian cancers bearing mt p53 (Soragni et al, Cancer Cell, 2016). We also reported that wt p53 AML cells responded to ReACp53 treatment (Zeng et al, Blood, 2016). ReACp53 eliminated misfolded p53, promoted its mitochondrial translocation and induced rapid apoptosis, suggesting that cytoplasmic misfolded wt p53 is a novel target in AML. MDM2 promotes p53 degradation, and inhibitors of MDM2 such as Nutlin derivatives are currently in trials for AML. These molecules inhibit p53 proteasomal degradation and result in p53-mediated apoptosis, as we demonstrated pre-clinically and in a Phase I trial of RG7112 in AML (Andreeff et al, Clin Cancer Res, 2015). p53 aggregation is initiated by protein misfolding, and progresses with increasing accumulation of misfolded p53. While p53 degradation is promoted by MDM2, binding of MDM2 to p53 causes p53 to misfold (Sasaki et al, J Biol Chem, 2007). This raises concerns about induction of p53 misfolding and consequent aggregation in tumors treated with MDM2 inhibitors, which could diminish therapeutic efficacy. We observed that levels of total and misfolded p53 and protein aggregation as identified by Proteostat positivity were MDM2 inhibitors dose- and time-dependent in wt p53 AML cells. This supports the hypothesis that MDM2 inhibition can cause not only p53 misfolding but also aggregation. Consequently, we show that adding a p53-aggregation inhibitor such as ReACp53 to an MDM2 inhibitor to limit p53 misfolding and aggregation results in increased cytotoxic activity in wt p53 AML. Co-aggregation of mt p53 with p63/p73 proteins carrying similar pro-aggregating segments has been reported (Xu et al, Nat Chem Biol, 2011). Next, we tested whether coaggregation could be an additional factor sequestering and inactivating wt p53. High levels of ΔNp73α, a tumor-promoting isoform of p73, can antagonize p53 function possibly through hetero-tetramer formation (Coutandin et al, Cell Death Differ, 2009), resulting in chemoresistance (Kazushi et al, Subcell Biochem, 2014). We hypothesize that upregulated ΔNp73α could constrain wt p53 through protein co-aggregation causing inactivation. Increased levels of misfolded p53 and protein aggregation were detected in both ΔNp73α-overexpressing HEK293T and MOLM13 (M13) cells. ΔNp73α-overexpressing M13 cells were resistant to MDM2 inhibitor-induced apoptosis compared to controls but sensitive to ReACp53. Treatment with Nutlin-derivatives (RG7388 or DS3032b) did not alter ΔNp73α levels but caused dose- and time-dependent increases in total and misfolded p53 and protein aggregation. HEK293T and M13 cells overexpressing ΔNp73α had higher levels of misfolded and aggregated p53, which we interpret as ΔNp73α providing a "seed" to accelerate p53 co-aggregation due to MDM2 inhibition. This suggests that ΔNp73α-overexpression conferred resistance to MDM2-mediated apoptosis that could be overcome by inhibition of p53 aggregation. Thus, combination of Nutlin derivatives and ReACp53 treatment exerted enhanced cytotoxicity in both cells lines. In conclusion, our data supports cytoplasmic, misfolded wt p53 as a novel target in AML and offers a rationale to combine therapeutic approaches supplementing MDM2 inhibition with p53 aggregation-targeting molecules to increase effectiveness. The model of wt p53 aggregation and coaggregation induced by MDM2 inhibition may apply to other cancer types. Disclosures Andreeff: Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership. Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Ishizawa:Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed.

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A Phase 1 Study of Milademetan in Combination with Quizartinib in Patients with Newly Diagnosed (ND) or Relapsed/Refractory (R/R) FLT3-ITD Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood-2019-122585 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1389-1389 ◽

Cited By ~ 1

Author(s):

Naval G. Daver ◽

Weiguo Zhang ◽

Richard Graydon ◽

Vikas K Dawra ◽

Jingdong Xie ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Phase 1 ◽

Wild Type ◽

Phase 1 Study ◽

Advisory Committees ◽

Once Daily ◽

Flt3 Inhibitor ◽

Equity Ownership ◽

Mdm2 Inhibitor

Background: FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutations occur in ≈ 25% of patients with AML and are associated with poor prognosis. Quizartinib is a once-daily, oral, highly potent and selective FLT3 inhibitor. In the phase 3 QuANTUM-R trial (NCT02039726; Cortes et al. Lancet Oncol 2019), quizartinib prolonged overall survival compared with salvage chemotherapy in patients with R/R FLT3-ITD AML. Murine double minute 2 (MDM2), an E3 ubiquitin ligase, negatively regulates the p53 tumor suppressor and has been shown to be upregulated in patients with AML; TP53 mutations in AML are infrequent except within complex karyotypes. Milademetan, a novel and specific MDM2 inhibitor, showed activity in an ongoing phase 1 trial in patients with AML or myelodysplastic syndromes (MDS) [DiNardo et al. ASH 2016, abstract 593]. Preclinical studies have shown that quizartinib plus milademetan may act synergistically to target FLT3-ITD and restore p53 activity in FLT3-ITD/TP53 wild-type AML [Andreeff et al. ASH 2018, abstract 2720]. Targeting MDM2 may restore p53 activity in cell signaling pathways altered by FLT3-ITD in patients with wild-type TP53 AML. Methods: This open-label, 2-part, phase 1 study (NCT03552029) evaluates quizartinib in combination with milademetan in patients with FLT3-ITD AML. Key inclusion criteria comprise a diagnosis of FLT3-ITD AML (de novo or secondary to MDS) and adequate renal, hepatic, and clotting functions. Key exclusion criteria include acute promyelocytic leukemia, prior treatment with a MDM2 inhibitor, QTcF interval > 450 ms, significant cardiovascular disease, and unresolved toxicities from prior therapies. Dose-escalation (part 1) comprises patients with R/R AML. In part 1, quizartinib will be administered once daily in 28-day cycles, at 3 proposed levels (30, 40, and 60 mg) with appropriate dose modifications based on QTcF monitoring and concomitant use of strong CYP3A inhibitors. Milademetan will be administered on days 1-14 of each 28-day cycle, at 3 proposed levels (90, 120, and 160 mg). Dose escalation will be guided by modified continual reassessment with overdose control. The primary objectives of part 1 are to evaluate the safety and tolerability, optimum dosing schedule, maximum tolerated dose (MTD), and recommended dosing for the expansion (RDE) cohort. Dose expansion (part 2) comprises a cohort of patients with R/R FLT3-ITD AML who have not received > 1 salvage therapy and not received > 1 prior FLT3 inhibitor, and a second cohort including ND patients with FLT3-ITD AML who are unfit for intensive chemotherapy. Patients in part 2 will be treated with quizartinib plus milademetan at the RDE doses identified in part 1. The objectives of part 2 are to confirm the safety and tolerability of quizartinib plus milademetan at RDE and identify the recommended phase 2 dose. Pharmacokinetics and preliminary assessment of efficacy are also being evaluated as secondary outcomes. Pharmacodynamic and biomarker assessments such as leukemic stem cell numbers, STAT5 downstream signaling, minimal residual disease measured by flow cytometry, and gene mutations will be evaluated as exploratory endpoints. Approximately 24 to 36 dose-limiting toxicity-evaluable patients are needed in part 1 to determine the MTDs and the RDE; approximately 40 patients per cohort will be treated at the RDE in part 2. This study is currently recruiting at multiple sites in the United States for part 1; recruitment for part 2 may be expanded to additional sites worldwide as necessary. Disclosures Daver: Jazz: Consultancy; Glycomimetics: Research Funding; Immunogen: Consultancy, Research Funding; Forty-Seven: Consultancy; Novartis: Consultancy, Research Funding; Servier: Research Funding; Karyopharm: Consultancy, Research Funding; Celgene: Consultancy; Abbvie: Consultancy, Research Funding; Agios: Consultancy; Daiichi Sankyo: Consultancy, Research Funding; Otsuka: Consultancy; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Hanmi Pharm Co., Ltd.: Research Funding; Genentech: Consultancy, Research Funding; Astellas: Consultancy; Incyte: Consultancy, Research Funding; Sunesis: Consultancy, Research Funding; NOHLA: Research Funding. Graydon:Daiichi Sankyo, Inc.: Employment. Dawra:Daiichi Sankyo, Inc.: Employment; Pfizer Inc: Employment. Xie:Daiichi Sankyo, Inc.: Employment. Kumar:Daiichi Sankyo, Inc.: Employment, Equity Ownership. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

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Synergistic Anti-Leukemic Activity with Combination of FLT3 Inhibitor Quizartinib and MDM2 Inhibitor Milademetan in FLT3-ITD Mutant/p53 Wild-Type Acute Myeloid Leukemia Models

Blood ◽

10.1182/blood-2018-99-115183 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2720-2720 ◽

Cited By ~ 3

Author(s):

Michael Andreeff ◽

Weiguo Zhang ◽

Prasanna Kumar ◽

Oleg Zernovak ◽

Naval G. Daver ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Combination Treatment ◽

Research Funding ◽

Single Agent ◽

Wild Type ◽

Advisory Committees ◽

Mouse Xenograft Model ◽

Equity Ownership ◽

Mdm2 Inhibitor

Abstract Background: MDM2, a negative regulator of the tumor suppressor p53, is overexpressed in several cancers including hematological malignancies. Disrupting the MDM2-p53 interaction represents an attractive approach to treat cancers expressing wild-type functional p53. Anticancer activity of small molecule MDM2 inhibitor milademetan (DS-3032b) has been demonstrated in preclinical studies and in a phase 1 trial in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome. Quizartinib is a highly selective and potent FLT3 inhibitor that has demonstrated single-agent activity and improvement in overall survival in a phase 3 clinical study in relapsed/refractory AML with FLT3-internal tandem duplication (FLT3-ITD) mutations. We present here the preclinical studies exploring the rationale and molecular basis for the combination of quizartinib and milademetan for the treatment of FLT3-ITD mutant/TP53 wild-type AML. Methods: We investigated the effect of quizartinib and milademetan combination on cell viability and apoptosis in established AML cell lines, including MV-4-11, MOLM-13 and MOLM-14, which harbor FLT3-ITD mutations and wild type TP53. Cells were treated with quizartinib and milademetan at specified concentrations; cell viability and caspase activation were determined by chemiluminescent assays, and annexin V positive fractions were determined by flow cytometry. We further investigated the effect of the combination of quizartinib and the murine specific MDM2 inhibitor DS-5272 in murine leukemia cell lines Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD+F691L and Ba/F3-FLT3-ITD+D835Y, which harbor FLT3-ITD, ITD plus F691L and ITD plus D835Y mutations, respectively. F691L or D835Y mutations are associated with resistance to FLT3-targeted AML therapy. In vivo efficacy of combination treatment was investigated in subcutaneous and intravenous xenograft models generated in male NOD/SCID mice inoculated with MOLM-13 and MV-4-11 human AML cells. Results: Combination treatment with milademetan (or DS-5272) and quizartinib demonstrated synergistic anti-leukemic activity compared to the respective single-agent treatments in FLT3 mutated and TP53 wild type cells. Combination indices (CIs) were 0.25 ± 0.06, 0.61 ± 0.03, 0.62 ± 0.06, 0.29 ± 0.004 and 0.50 ± 0.03, respectively, in MV-4-11, MOLM-13, MOLM-14, Ba/F3-FLT3-ITD+F691L and D835Y cell lines, all of which harbor FLT3-ITD or ITD plus TKD point mutations. The combination regimen triggered synergistic pro-apoptotic effect in a p53-dependent manner as shown by annexin-V staining and caspase 3/7 assays. Mechanistically, the combination treatment resulted in significant suppression of phospho-FLT3, phospho-ERK and phospho-AKT and anti-apoptotic Bcl2 family proteins (eg, Mcl-1), as well as up-regulation of p53, p21 and pro-apoptotic protein PUMA, compared to single agent treatments. Of note, the combination regimen also exerted a synergistic pro-apoptotic effect on venetoclax (BCL-2 inhibitor)-resistant MOLM-13 cells (CI: 0.39 ± 0.04) through profound suppression of Mcl-1. In an in vivo study using the MOLM-13 subcutaneous mouse xenograft model, quizartinib at 0.5 and 1 mg/kg and milademetan at 25 and 50 mg/kg demonstrated a significant tumor growth inhibition compared with vehicle treatment or respective single-agent treatments. In MV-4-11 intravenous mouse xenograft model, the combination of quizartinib plus milademetan showed a significantly prolonged survival, with no animal death in the combination group during the study period, compared to respective single agent treatments and untreated control (Figure). Conclusion: Synergistic anti-leukemic activity was observed for quizartinib plus milademetan combination treatment in preclinical AML models. A phase I clinical trial of quizartinib/milademetan combination therapy in patients with FLT3-ITD mutant AML is underway. Figure. Effects of quizartinib, milademetan and their combination on survival of mice intravenously inoculated with human MV-4-11 AML cells Disclosures Andreeff: Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Oncolyze: Equity Ownership; Astra Zeneca: Research Funding; Reata: Equity Ownership; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; SentiBio: Equity Ownership. Kumar:Daiichi Sankyo: Employment, Equity Ownership. Zernovak:Daiichi Sankyo: Employment, Equity Ownership. Daver:Pfizer: Research Funding; ImmunoGen: Consultancy; Otsuka: Consultancy; Karyopharm: Research Funding; Alexion: Consultancy; ARIAD: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Karyopharm: Consultancy; Novartis: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Kiromic: Research Funding; Sunesis: Research Funding; Incyte: Consultancy; Pfizer: Consultancy; Sunesis: Consultancy. Isoyama:Daiichi SANKYO CO., LTD.: Employment. Iwanaga:Daiichi Sankyo Co., Ltd.: Employment. Togashi:Daiichi SANKYO CO., LTD.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment.

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Clonal Expansion of Mutant p53 Clones By MDM2 Inhibition in Acute Myeloid Leukemias

Blood ◽

10.1182/blood-2020-142794 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

Yuki Nishida ◽

Rafael Heinz Montoya ◽

Kiyomi Morita ◽

Tomoyuki Tanaka ◽

Feng Wang ◽

...

Keyword(s):

Single Cell ◽

Board Of Directors ◽

Research Funding ◽

Drug Exposure ◽

Single Agent ◽

Wild Type ◽

Advisory Committees ◽

Tp53 Mutations ◽

Mdm2 Inhibitor ◽

Acute Myeloid

MDM2 inhibition by small molecules as a means of restoring p53 function has shown clinical activity against acute myeloid leukemia (AML) (Andreeff, Clin Cancer Res 2015). However, we and others have found increased variant allele frequencies (VAFs) of TP53 mutations in AML cells after treatment with MDM2 inhibitors, either as monotherapy or in combination with other agents (Daver ASH 2019), which suggests that MDM2 inhibition selects preexisting clones or generates de novo clones with TP53 mutations. We performed a long-term culture of AML cells (MOLM-13, an AML cell line with wild-type p53 and FLT3-ITD) treated with increasing concentrations of an MDM2 inhibitor idasanutlin (up to 320 nM, less than 10% concentration of Cmax) (Selleck). We obtained MDM2 inhibitor-resistant (R) AML cells after 96 days of the drug exposure and found that the resistant cells harbor hotspot TP53 p.R248W (R248W) mutation. We next isolated single cell clones from MOLM-13 R cells by limiting dilution, and obtained twelve subclones (subclones #1-12 in order of development). All clones carried the same R248W mutation. To determine clonal patterns of these cells, we performed single cell DNA sequencing (scDNAseq) of MOLM-13 parental, R and subclone #1 and #2 (SC1 and SC2) cells using the MissionBio Tapestry system covering 125 amplicons of 19 genes frequently mutated in AML. scDNAseq identified FLT3-ITD mutations in all cells analyzed, as expected. In the parental cells we identified only 0.02% cells (1/ 5,240) with the identical R248W mutation found in MOLM-13 R cells. MOLM-13 R cells had only 0.6% of wild-type TP53 cells, 51% carrying R248W only, and 43% R248W/R213* mutation (R248W/R213*). SC1 and SC2 cells had 1% and 99% of R248W and R248W/R213* clones, respectively (Fig.1). Seven other mutations were detected by scDNAseq. Results suggest that MDM2 inhibition can accelerate the selection of TP53-mutant AML cells in vitro. Of note, the parental cells had remained mostly p53 wild-type, where the subclone with mutant R248W did not have a growth advantage over other cells. Next we analyzed patient samples enrolled in the phase 1 clinical trial (NCT02319369) for the MDM2 inhibitor milademetan (DS-3032b; Daiichi-Sankyo) in relapsed/refractory AML or high-risk MDS patients. Fifty seven patients were treated with single agent milademetan in the study. All but one patient had wild-type TP53 as determined by NGS at baseline. One patient (1.8%) had a TP53 p.R213* mutation at baseline with VAF of 91%. Four patients (7%) developed different TP53 mutations (R248Q, R248W, P250fs, V122fs and V274L), with increasing VAFs that were not detected at baseline to 19% average, ranging from 11% to 28% post treatment, One patient (anonymized ID 1001-1005) developed both, R248Q and R248W mutations, detected at cycle 2 day1 (C2D1, day 29). The pre-existing R213* mutation detected in one patient persisted with increased VAF after treatment (91% to 100%). To detect p53 mutations with higher sensitivity than NGS, we performed droplet digital PCR (ddPCR) for R248W/Q and R273H in samples from two patients. ddPCR detected 0.46% and 0.62% of R248Q and R248W mutations, respectively, at baseline, which were not detected by NGS, with increased VAFs of 18.2 and 27.6% at C2D1, respectively. ddPCR detected additional R273H mutations with VAFs of 0.08% and 0.18% at baseline, and 2.2% and 2.6% in these patients at C2D1, respectively. Conclusion: Data suggest that MDM2 inhibition selects rare AML subpopulations with TP53 mutations and careful monitoring of patients treated with MDM2 inhibitors with sensitive methods to detect TP53 mutant clones is warranted. This finding also points to the need to develop strategies to prevent/suppress these clones early on. Disclosures Kumar: Daiichi-Sankyo Inc.: Current Employment. Patel:Daiichi-Sankyo Inc.: Current Employment. Dos Santos:Daiichi Sankyo, Inc.: Current Employment. DiNardo:Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Novartis: Consultancy; ImmuneOnc: Honoraria; Calithera: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Notable Labs: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Research Funding; MedImmune: Honoraria; Jazz: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Syros: Honoraria. Andreeff:Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding.

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EZH2 Mutations and Impact on Clinical Outcome Analyzed in 1604 Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114421 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1528-1528

Author(s):

Sebastian Stasik ◽

Jan Moritz Middeke ◽

Michael Kramer ◽

Christoph Rollig ◽

Alwin Krämer ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Mutational Status ◽

Equity Ownership ◽

Acute Myeloid

Abstract Purpose: The enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase and key epigenetic regulator involved in transcriptional repression and embryonic development. Loss of EZH2 activity by inactivating mutations is associated with poor prognosis in myeloid malignancies such as MDS. More recently, EZH2 inactivation was shown to induce chemoresistance in acute myeloid leukemia (AML) (Göllner et al., 2017). Data on the frequency and prognostic role of EZH2-mutations in AML are rare and mostly confined to smaller cohorts. To investigate the prevalence and prognostic impact of this alteration in more detail, we analyzed a large cohort of AML patients (n = 1604) for EZH2 mutations. Patients and Methods: All patients analyzed had newly diagnosed AML, were registered in clinical protocols of the Study Alliance Leukemia (SAL) (AML96, AML2003 or AML60+, SORAML) and had available material at diagnosis. Screening for EZH2 mutations and associated alterations was done using Next-Generation Sequencing (NGS) (TruSight Myeloid Sequencing Panel, Illumina) on an Illumina MiSeq-system using bone marrow or peripheral blood. Detection was conducted with a defined cut-off of 5% variant allele frequency (VAF). All samples below the predefined threshold were classified as EZH2 wild type (wt). Patient clinical characteristics and co-mutations were analyzed according to the mutational status. Furthermore, multivariate analysis was used to identify the impact of EZH2 mutations on outcome. Results: EZH2-mutations were found in 63 of 1604 (4%) patients, with a median VAF of 44% (range 6-97%; median coverage 3077x). Mutations were detected within several exons (2-6; 8-12; 14-20) with highest frequencies in exons 17 and 18 (29%). The majority of detected mutations (71% missense and 29% nonsense/frameshift) were single nucleotide variants (SNVs) (87%), followed by small indel mutations. Descriptive statistics of clinical parameters and associated co-mutations revealed significant differences between EZH2-mut and -wt patients. At diagnosis, patients with EZH2 mutations were significantly older (median age 59 yrs) than EZH2-wt patients (median 56 yrs; p=0.044). In addition, significantly fewer EZH2-mut patients (71%) were diagnosed with de novo AML compared to EZH2-wt patients (84%; p=0.036). Accordingly, EZH2-mut patients had a higher rate of secondary acute myeloid leukemia (sAML) (21%), evolving from prior MDS or after prior chemotherapy (tAML) (8%; p=0.036). Also, bone marrow (and blood) blast counts differed between the two groups (EZH2-mut patients had significantly lower BM and PB blast counts; p=0.013). In contrast, no differences were observed for WBC counts, karyotype, ECOG performance status and ELN-2017 risk category compared to EZH2-wt patients. Based on cytogenetics according to the 2017 ELN criteria, 35% of EZH2-mut patients were categorized with favorable risk, 28% had intermediate and 37% adverse risk. No association was seen with -7/7q-. In the group of EZH2-mut AML patients, significantly higher rates of co-mutations were detected in RUNX1 (25%), ASXL1 (22%) and NRAS (25%) compared to EZH2-wt patients (with 10%; 8% and 15%, respectively). Vice versa, concomitant mutations in NPM1 were (non-significantly) more common in EZH2-wt patients (33%) vs EZH2-mut patients (21%). For other frequently mutated genes in AML there was no major difference between EZH2-mut and -wt patients, e.g. FLT3ITD (13%), FLT3TKD (10%) and CEBPA (24%), as well as genes encoding epigenetic modifiers, namely, DNMT3A (21%), IDH1/2 (11/14%), and TET2 (21%). The correlation of EZH2 mutational status with clinical outcomes showed no effect of EZH2 mutations on the rate of complete remission (CR), relapse free survival (RFS) and overall survival (OS) (with a median OS of 18.4 and 17.1 months for EZH2-mut and -wt patients, respectively) in the univariate analyses. Likewise, the multivariate analysis with clinical variable such as age, cytogenetics and WBC using Cox proportional hazard regression, revealed that EZH2 mutations were not an independent risk factor for OS or RFS. Conclusion EZH mutations are recurrent alterations in patients with AML. The association with certain clinical factors and typical mutations such as RUNX1 and ASXL1 points to the fact that these mutations are associated with secondary AML. Our data do not indicate that EZH2 mutations represent an independent prognostic factor. Disclosures Middeke: Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Rollig:Bayer: Research Funding; Janssen: Research Funding. Scholl:Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Abbivie: Other: Travel support; Alexion: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Deutsche Krebshilfe: Research Funding; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Hochhaus:Pfizer: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Takeda: Research Funding. Brümmendorf:Janssen: Consultancy; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Merck: Consultancy; Pfizer: Consultancy, Research Funding. Burchert:AOP Orphan: Honoraria, Research Funding; Bayer: Research Funding; Pfizer: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Novartis: Research Funding. Krause:Novartis: Research Funding. Hänel:Amgen: Honoraria; Roche: Honoraria; Takeda: Honoraria; Novartis: Honoraria. Platzbecker:Celgene: Research Funding. Mayer:Eisai: Research Funding; Novartis: Research Funding; Roche: Research Funding; Johnson & Johnson: Research Funding; Affimed: Research Funding. Serve:Bayer: Research Funding. Ehninger:Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; Bayer: Research Funding; GEMoaB Monoclonals GmbH: Employment, Equity Ownership. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

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Genetic Dissection of p53 Driven Senescence of Bone Marrow Mesenchymal Cells in Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-117585 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2625-2625

Author(s):

Rasoul Pourebrahim ◽

Peter P. Ruvolo ◽

Steven M. Kornblau ◽

Carlos E. Bueso-Ramos ◽

Michael Andreeff

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Bone Marrow Mscs ◽

Advisory Committees ◽

Equity Ownership ◽

Bone Marrow Mesenchymal Cells ◽

Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a genetically heterogeneous malignancy characterized by bone marrow infiltration of abnormally proliferating leukemic blasts which results in fatal anemia, bleeding and infectious complications due to compromised normal hematopoiesis. Patients with complete remission (CR) but incomplete blood cell count recovery (CRi) have significantly shorter survival compared to CR patients. Although there is a correlation between CRi and minimal residual disease (MRD), the two variables were shown to be independent risk factors for relapse development (1). The mechanism by which AML induces bone marrow failure in patients is largely unknown. Here, we demonstrate that AML derived MSCs highly express p53 and p21 proteins and are more senescent compared to their normal age-matched controls as demonstrated by high β-galactosidase staining (figure 1. A, B&C). Emerging evidence indicates that the aging of endosteal niche cells results in lower reconstitution potential of hematopoietic stem cells (2). To functionally evaluate the effects of AML on bone marrow MSCs, we utilized a murine leukemia model of the AML microenvironment. We transplanted Osx-Cre;mTmG mice with AML cells and compared the senescence of MSCs in normal bone marrow (Figure 1.D) with AML (Figure 1.E). Consistent with our initial findings in human, AML strongly induced senescence of osteoblasts. This suggests that AML suppresses normal hematopoiesis by inducing senescence in the hematopoietic niche. To address the role of p53 signaling in senescence of MSCs we generated a traceable conditional p53 gain/loss model specifically in bone marrow MSCs using Osx-Cre;mTmG; Mdm2fl/+ and Osx-Cre;mTmG;p53fl/fl mice respectively (Figure 1.F). Deletion of p53 in bone marrow MSCs resulted in an increased population of osteoblasts (GFP+) in Osx-Cre;mTmG;p53fl/fl mice in comparison to Osx-Cre;mTmG mice suggesting that p53 loss in osteoblasts inhibits senescence of osteoblasts. In order to evaluate p53 activity after recombination of p53fl alleles in the osteoblasts, we isolated MSCs from bone marrows and analyzed the expression of p21.P21 was significantly down regulated in osteoblasts (GFP+) derived from Osx-Cre;mTmG;p53fl/fl mice whereas its expression in the hematopoietic cells from same tissue (tdTomato+) remained comparable to p53 wild type suggesting that p21 as the master regulator of senescence is regulated by p53 in bone marrow mesenchymal cells. To evaluate the effect of p53 loss in osteoblasts and its impact on hematopoietic cells, we isolated the GFP+ cells (osteoblasts) and RFP + cells (hematopoietic) by FACS. Senescent cells, non-cell autonomously, modulate the bone marrow microenvironment through the senescence-associated secretory phenotype (SASP). We analyzed the expression of fifteen SASP cytokines by QPCR. Deletion of p53 in bone marrow mesenchymal cells strongly abrogated the expression of several SASP cytokines. Interestingly several Notch target genes such as Hey1 and Hey2 were highly induced in MSCs following p53 deletion suggesting a role for Notch signaling in hematopoietic failure following AML induced MSCs senescence. Our data suggest that AML induces senescence of endosteal niche resulting in hematopoietic failure. These findings contribute to our understanding of the role of p53 in leukemia MSCs and could have broad translational significance for the treatment of hematopoietic failure in patients with AML.Chen X, et al. (2015) Relation of clinical response and minimal residual disease and their prognostic impact on outcome in acute myeloid leukemia. J Clin Oncol 33(11):1258-1264.Li J, et al. (2018) Murine hematopoietic stem cell reconstitution potential is maintained by osteopontin during aging. Sci Rep 8(1):2833. Disclosures Andreeff: Astra Zeneca: Research Funding; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncolyze: Equity Ownership; Jazz Pharma: Consultancy; Reata: Equity Ownership.

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Acute Myeloid Leukemia Expands Osteoprogenitor Rich Niche in the Bone Marrow but Resorbs Mature Bone Causing Osteopenia/Osteoporosis in Animal Models

Blood ◽

10.1182/blood-2018-99-117258 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 86-86 ◽

Cited By ~ 1

Author(s):

Bin Yuan ◽

Stanley Ly ◽

Khoa Nguyen ◽

Vivien Tran ◽

Kiersten Maldonado ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Bone Surface ◽

Advisory Committees ◽

Osteoprogenitor Cells ◽

Equity Ownership ◽

Acute Myeloid ◽

Mature Bone

Abstract Acute myeloid leukemia (AML) is one of the most aggressive hematological malignancy that originates in the bone marrow (BM). Despite advances in the molecular characterization of AML, factors regulating its progression are still not known. Among several BM niches that support AML growth in the BM, the osteogenic niche has gained attention in recent years owing to its potential role in leukemogenesis. Genetic alterations in osteoprogenitor cells have been shown to induce myeloid leukemia in mouse models. We reported recently that AML cells induce osteogenic differentiation in mesenchymal stromal cells (MSCs) in the BM to facilitate faster AML engraftment in mice (Battula et al., JCI Insight, 2017). However specifics of this osteogenic niche generated by AML are not known. Here we hypothesize that AML expands osteo-progenitor rich niche in the BM, but that the mature bone is reduced. To determine the type of AML-induced osteo-lineage differentiation in the BM, we generated transgenic reporter mice by crossing Osx-CreERt2 mice with Ocn-GFP; ROSA-tdTomato mice. The resulting triple transgenic mice has the genotype of Osx-CreERt2;Ocn-GFP;ROSA-tdTomato. In these mice the tdTomato (red) positive cells represents osteo-lineage cells that originate from Osterix expressing (Osx+) cells, whereas a GFP+ cell represents an osteocalcin-expressing (Ocn+) mature osteoblast. Seven day old triple transgenic mice were injected with tamoxifen to activate Osx-CreERT2 to mark the Osx+ cells with tomato reporter. To investigate the osteogenic cell type that is induced by AML cells in the bone marrow, we implanted murine AML cells with MLL-ENL fusion proteins into Osx-CreERt2;Ocn-GFP;ROSA-tdTomato mice. Three weeks after implantation of AML cells, the femurs and tibia of these mice were dissected and subjected to histological evaluation using fluorescence microscopy. In control BM without AML, the GFP+ (Ocn+) cells were found in the trabecular bone surface as well as the periosteum of the bone, whereas the tdTomato+ (Osx+)cells were found in the marrow and the bone matrix; this suggests that some of the osteocytes originated from tamoxifen-induced Osx+ osteoprogenitor cells. Interestigly, in mice implanted with AML cells, we found a 3-4 fold increase in Osx+ cells in the marrow compared to normal BM (Fig 1A). However, the number of GFP+ cells on the endosteum and trabecular bone surface was reduced, suggesting that AML cells might expand osteoprogenitor cells but not fully differentiated mature osteoblasts. Next, to investigate whether AML cells affect the mature bone, AML PDX cells developed in our laboratory were implanted into NSG mice. The PDX models usually take 12-14 weeks to achieve >90% engraftment in the peripheral blood which provides ample time to observe alterations in bone composition. At this stage, the mice were subjected to computed tomography imaging to measure bone architecture, volume (BV), mineral density (BMD) and bone volume fraction (BVF). Interestingly, we observed large bone cavities close to epiphysis and metaphysis areas in the femur and tibia of mice with AML (Fig 1B). In addtion, BMD and BVF in these mice were reduced by 20-30% compared to control mice without leukemia. To validate the bone resorption in these mice, bone histomorphometric analysis was performed on femurs and tibias from mice with and without AML. Masson-Goldner's Trichrome staining revealed a 5- to 10-fold decrease in the trabecular and cortical bone thickness in AML femurs compared to normal femurs. Moreover, measurements of osteoclast activation by tartrate-resistant acidic phosphatase (TRAP) revealed positive staining for osteoclasts on the endosteal surface and massive bone resorption in AML bone compared to normal bone. Mechanistic studies showed that AML cells inhibit osteoprotegerin (OPG) ~10 fold in MSCs, a factor that inhibits the RNAK ligand which in turn activates osteoclasts that breakdown the bone. In conclusion, our data suggest that bone homeostasis is dysregulated in AML by induction of osteogenic and osteolytic activities simultaneously. AML cells induce an osteoprogenitor niche but also activate osteoclasts resulting in osteopenia/osteoporosis in mouse models. In-depth analysis of bone remodeling in AML patients could result in new insights into the pathobiology of the disease and provide therapeutic avenues for AML. Disclosures Andreeff: Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Celgene: Consultancy; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; SentiBio: Equity Ownership; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership. Battula:United Therapeutics Inc.: Patents & Royalties, Research Funding.

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