Inhibition of MDM2 Improves the Therapeutic Effect of Hypomethylating Agents in Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML)

Abstract Hypomethylating agents (HMA) are the current standard care for MDS and CMML. However, a large proportion of patients experience HMA treatment failure that is associated with poor prognosis. Novel treatments are urgently needed for these diseases. We performed next generation sequencing (NGS) based mutation profiling that targeted the 81 most frequently mutated genes in baseline bone marrow (BM) mononuclear cells (MNCs) from patients with MDS and CMML (N=83) prior to HMA treatment. Analysis of impact of the mutations on outcomes of HMA treatment indicated that patients with TP53 mutations (N=22) had significantly worse overall survival (HR = 3.29; P < 0.05; q < 0.05). This result is consistent with previous reports and suggests that strengthening the activity of wild type (WT) TP53, through strategies such as inhibition of TP53 negative regulator MDM2, may improve therapeutic efficacy of HMAs in patients with MDS and CMML. To test this hypothesis, we evaluated in vitro the effects of the combination of the MDM2 inhibitor DS-3032b and azacytidine (AZA). Due to the dearth of MDS or CMML cell lines, initial analysis was performed in the TP53 WT AML cell line MOLM13. Results indicated that DS-3032b and AZA combination induced a synergistic decrease in cell survival (coefficient of drug interaction=0.65), accompanied with increased apoptosis and CDKN1A1 (P21) RNA expression. No combination survival effect was observed in the TP53 mutant AML cell lines SKM1 or TF1. We next evaluated in vivo combination therapeutic effects of the MDM2 inhibitor DS-5272 and AZA in a Tet2-knockout (KO) (Tet2flox/flox/Vav-Cre) mouse model. Prior work indicated that Tet2-KO mice had an MDS/CMML-like phenotype and a persistent long-term (LT) BM repopulating activity that was resistant to AZA. DS-5272 and AZA were administered to mice sequentially: DS-5272 (50 mg/kg) was administered orally for 5 days, followed by daily AZA (2.5 mg/kg, i.p.) for a 7 days. DS-5272 was also administered on the 1st and 3rd days during AZA treatment. After a 2-week gap, the treatment cycle was repeated once. Following the combination treatment, Tet2-KO mice showed significantly reduced monocytosis (P<0.05) and increased platelet counts (P<0.001) in peripheral blood (PB), and significant reductions in Gr1+/CD11b1+ myeloid cells (P<0.001), common myeloid progenitors (CMPs) (P<0.005), and Lin-/Sca1+/c-Kit1+ cells (LSKs) (P<0.01) in BM. Next, we evaluated the impact of treatment on LT BM repopulation activity. CD45.2+ BM cells isolated from drug-treated Tet2-KO mice were mixed with BM cells from CD45.1 WT mice and then transplanted into lethally irradiated CD45.1 WT recipient mice. Significantly lower (P<0.05) CD45.2 chimerism was observed in PB of the recipients transplanted by BM cells from DS-5272 and AZA combination treated Tet2-KO mice compared to recipients that received cells from vehicle control and mono-agent treated Tet2-KO mice, starting from 4 months post-transplantation (Fig.1A). Lower CD45.2 chimerism was also observed in the BM LSK population (P<0.001) of the same recipients at the endpoint of 6 months post-transplantation. To study the molecular mechanisms underlying the combinational therapeutic effects of the MDM2 inhibitor and AZA observed in the Tet2-KO mice, we performed RNA-seq on BM LSK cells isolated from drug-treated mice. Compared to LSKs from vehicle control mice, over 800 genes with significantly altered RNA expression (>1.5 fold; FDR<0.05) were identified in the LSKs from DS-5272 and AZA combination treated Tet2-KO mice, whereas 110 and 476 genes with altered RNA expression were identified in the LSKs of DS-5272 or AZA treated Tet2-KO mice respectively. REACTOME pathway analysis identified 263 pathways that were significantly (FDR<0.25) and uniquely activated in the BM LSKs from mice treated with DS-5272 and AZA combination. Many of these pathways were associated with TP53 stability, TP53 activity, and TP53 regulated apoptosis and cell cycle (Fig.1B), suggesting a coordination between MDM2 inhibition and HMA treatment to activate TP53 in BM HSPCs. In conclusion, our work provides proof-of-concept evidence that combining an MDM2 inhibitor with AZA can improve the therapeutic efficacy of AZA in MDS and CMML, through mechanisms including synergistic activation of TP53 in BM HSPCs and inhibition of LT BM repopulating activity. This concept should be further evaluated through pre-clinical studies and clinical trials. Figure 1 Figure 1. Disclosures Wei: Daiichi Sanko: Research Funding. Daver: Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Hanmi: Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Genentech: Consultancy, Research Funding; Sevier: Consultancy, Research Funding; Novimmune: Research Funding; FATE Therapeutics: Research Funding; Trovagene: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis: Consultancy; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding.

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Exome Sequencing of Aggressive Natural Killer Cell Leukemia and Drug Profiling Highlight Candidate Driver Pathways in Malignant Natural Killer Cells

Blood ◽

10.1182/blood.v126.23.700.700 ◽

2015 ◽

Vol 126 (23) ◽

pp. 700-700

Author(s):

Olli Dufva ◽

Tiina Kelkka ◽

Shady Awad ◽

Nodoka Sekiguchi ◽

Heikki Kuusanmäki ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Exome Sequencing ◽

Cell Lines ◽

Hierarchical Clustering ◽

Research Funding ◽

Drug Sensitivity ◽

Somatic Mutations ◽

Nk Cell ◽

Negative Regulator

Abstract Background Natural killer (NK) cell malignancies are rare lymphoid neoplasms characterized by aggressive clinical behavior and poor treatment outcomes. Clinically they are classified as extranodal NK/T-cell lymphoma, nasal type (NKTCL) and aggressive NK cell leukemia (ANKL). Both subtypes are almost invariably associated with Epstein-Barr virus (EBV). Recently, genomic studies in NKTCL have identified recurrent somatic mutations in JAK-STAT pathway molecules STAT3 and STAT5b as well as in the RNA helicase gene DDX3X in addition to previously detected chromosomal aberrations. Here, we identified somatic mutations in 4 cases of ANKL in order to understand whether these entities share common alterations at the molecular level. To further establish common patterns of deregulated oncogenic signaling pathways operating in malignant NK cells, we performed drug sensitivity profiling using NK cell lines representing ANKL, NKTCL and other malignant NK cell proliferations. We aimed to identify sensitivities to agents that selectively target components of pathways required for survival of malignant NK cells in an unbiased manner. Methods Exome sequencing was performed on peripheral blood or bone marrow of ANKL patients using the NK cell negative fraction or other healthy tissue as control. Profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising 461 approved and investigational oncology drugs. The NK cell lines KAI3, KHYG-1, NKL, NK-YS, NK-92, SNK-6 and YT and IL-2-stimulated and resting NK cells from healthy donors were used as sample material. All drugs were tested on a 384-well format in 5 different concentrations over a 10,000-fold concentration range for 72 h and cell viability was measured. A Drug Sensitivity Score (DSS) was calculated for each drug using normalized dose response curve values. Results The ANKL patients displayed mutations in genes reported as recurrently mutated in NKTCL, such as FAS, TP53, NRAS, STAT3 and DDX3X. Additionally, novel alterations in genes previously implicated in the pathogenesis of NKTCL were detected. These included an inactivating mutation in INPP5D (SHIP), a negative regulator of the PI3K/mTOR pathway and a missense mutation in PTPRK, a negative regulator of STAT3 activation. Interestingly, the total number of nonsilent somatic mutations in 3 out of 4 ANKL patients (97, 82 and 45) was remarkably high compared to other hematological malignancies analyzed in our variant calling pipeline. Analysis of drug sensitivities in NK cell lines showed a close correlation between all cell lines and a markedly higher correlation with those of IL-2 stimulated than resting healthy NK cells, suggesting that malignant NK cells may share a common drug response pattern. Furthermore, in an unsupervised hierarchical clustering the NK cell lines formed a distinct group from other leukemia cell lines tested (Fig. A). Among pathway-selective compounds (namely, kinase inhibitors and rapalogs), the drugs most selective for malignant NK cells fell into two major categories: PI3K/mTOR inhibitors (e.g. temsirolimus, buparlisib) and inhibitors of aurora and polo-like kinases such as rigosertib and GSK-461364 (Fig. B). JAK inhibitors (e.g. ruxolitinib, gandotinib) and CDK inhibitors (e.g. dinaciclib) showed strong efficacy in both malignant NK cells and IL-2 activated healthy NK cells. Conclusions Our exome sequencing results suggest that candidate driver alterations affecting similar signaling pathways underlie the pathogenesis of ANKL as has been reported in NKTCL. Drug sensitivity profiling highlights the PI3K/mTOR pathway as a potential major driver of malignant NK cell proliferation, whereas JAK-STAT signaling appears to be essential in both healthy and malignant NK cells. Components of these pathways harbored mutations in our small cohort of ANKL patients and have been shown to be deregulated by mutations or other mechanisms in previous studies, underlining their importance as putative drivers. The systematic large-scale characterization of drug responses also identified these pathways as potential targets for novel therapy strategies in NK cell malignancies. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Figure 1. (A) Unsupervised hierarchical clustering based on drug sensitivity scores (DSS) of NK, AML, CML and T-ALL cell lines. (B) Scatter plot comparing DSS of malignant NK cell lines (average) and healthy IL-2 stimulated NK cells. Disclosures Mustjoki: Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

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BI-1206, a Monoclonal Antibody Against Fcγriib, Showed Superior Anti-Tumor Activity in an Ibrutinib-Venetoclax Dual Resistant PDX Model in Mantle Cell Lymphoma

Blood ◽

10.1182/blood-2019-126957 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2863-2863

Author(s):

Changying Jiang ◽

Yang Liu ◽

Joseph Mitchell McIntosh ◽

Angela Leeming ◽

Rongjia Zhang ◽

...

Keyword(s):

Cell Surface ◽

Cell Lines ◽

Therapeutic Efficacy ◽

Research Funding ◽

Single Agent ◽

Flow Cytometry Analysis ◽

Immune Effector ◽

Pdx Model ◽

Mantle Cell

Introduction: Mantle cell lymphoma (MCL) is a rare but aggressive non-Hodgkin lymphoma (NHL). CD20 antibodies (e.g. rituximab), BTK inhibitors (e.g. ibrutinib and acalabrutinib), and BCL-2 inhibitors (e.g. venetoclax) alone or in combination have shown great anti-MCL efficacy. However, primary and acquired resistance to one or multiple therapies commonly occurs, resulting in poor clinical outcome. Overcoming such mechanisms of resistance holds promise to significantly improve survival, meeting a significant medical need for patients with refractory/relapsed MCL. Recent studies showed Fc gamma receptors (FcγRs) play important roles in controlling therapeutic efficacy. FcgRIIB (CD32B), the inhibitory FcγR, negatively controls antibody efficacy through distinct inhibitory mechanisms in immune effector cells and lymphoma cells, respectively. When expressed on leukemic or lymphoma cells, FcgRIIB promotes rituximab internalization and removal from the tumor cell surface, resulting in reduced immune effector cell activation and ultimately decreased in vivo therapeutic efficacy. We recently developed antagonistic antibodies to FcgRIIB and demonstrated that these antibodies blocked rituximab internalization and helped prevent and overcome rituximab resistance in a PDX model of CLL. In this study, we investigated the expression of FcgRIIB in MCL cell lines and primary patient MCL samples, and we assessed the in vivo efficacy of BI-1206, a monoclonal antibody against FcgRIIB, in MCL PDX models. Methods: Flow cytometry analysis was performed to examine the cell surface expression of FcgRIIB in MCL cell lines (n=8) and primary patient MCL samples (n=27). An orthotopic patient-derived xenograft (PDX) model was established from a MCL patient with dual resistance to ibrutinib-venetoclax. In the first mouse cohort, single-agent ibrutinib, venetoclax, or vehicle control were administrated in mice carrying the orthotopic PDX model to assess their in vivo efficacies. In the second mouse cohort, mice were treated with vehicle, single agent BI1206, rituximab plus lenalidomide, or a combination of BI-1206, rituximab, and lenalidomide (triple combination) to assess their in vivo efficacies in the same PDX model. Results: Flow cytometry analysis showed that all 8 MCL cell lines and all 27 primary patient MCL samples expressed high levels of FcgRIIB, highlighting the potential importance of FcgRIIB on the control of therapeutic efficacy in MCL. In the first mouse cohort, we validated the ibrutinib and venetoclax resistance in the PDX model established from a MCL patient with resistance to both therapies. In the second mouse cohort, single agent BI-1206 (10 mg/kg, twice a week) potently diminished PDX growth in spleen, liver, bone marrow, and peripheral blood (Figure 1). Treatment with rituximab (10 mg/kg, twice per week) plus lenalidomide (50mg/kg, daily) or the triple combination showed similar potency (Figure 1). To investigate whether BI-1206 mediates boosting of rituximab-based targeted drug therapies, and/or overcoming of resistance to such therapies, a follow-up experiment with revised treatment setting using the same PDX model or an alternative CD20/FcγRIIb co-expressing PDX model is currently under investigation. Conclusions: All MCL cell lines and all primary MCL cells tested highly express FcγRIIb on the tumor cell surface. Single agent BI-1206 has potent anti-MCL activity in the FcγRIIb-expressing MCL PDX model to overcome ibrutinib-venetoclax dual resistance. Our data suggests FcγRIIb may be an important target for anti-MCL therapies. Disclosures Wang: Guidepoint Global: Consultancy; Kite Pharma: Consultancy, Research Funding; Acerta Pharma: Consultancy, Research Funding; MoreHealth: Consultancy, Equity Ownership; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Pharmacyclics: Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; BioInvent: Consultancy, Research Funding; Aviara: Research Funding; Juno Therapeutics: Research Funding; Celgene: Honoraria, Research Funding; Loxo Oncology: Research Funding; VelosBio: Research Funding; Dava Oncology: Honoraria.

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Discovery of Predictive Gene Signatures for Tumor Sensitivity to MDM2 Inhibition in Development of a Novel MDM2 Inhibitor DS-3032b

Blood ◽

10.1182/blood.v128.22.2893.2893 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2893-2893 ◽

Cited By ~ 1

Author(s):

Jo Ishizawa ◽

Kenji Nakamaru ◽

Takahiko Seki ◽

Koichi Tazaki ◽

Kensuke Kojima ◽

...

Keyword(s):

Cell Lines ◽

Research Funding ◽

Tp53 Mutation ◽

Gene Signature ◽

Advisory Board ◽

Wild Type ◽

Mutation Status ◽

Gene Signatures ◽

Gene Set ◽

Mdm2 Inhibitor

Abstract Development of MDM2 inhibitors enabled successful induction of p53-mediated apoptosis in tumor cells without a risk of DNA damage. Early clinical trials of MDM2 inhibitors demonstrated proof-of-concept (Andreeff et al., Clin Can Res, 2015). However, a clinical challenge is that not all the tumors bearing wild-type TP53 are sensitive to MDM2 inhibition. We here discovered novel gene profiling-based algorithms for predicting tumor sensitivity to MDM2 inhibition, using DS-3032b, a novel potent MDM2 inhibitor, which is currently in early clinical trials. In vitro inhibitory effects of DS-3032b on MDM2-p53 interaction was demonstrated using the homogeneous time resolved fluorescence (HTRF) assay (IC50 5.57 nM). DS-3032b treatment (30-1000 nM) indeed increased p53 protein in a dose-dependent manner, and also the p53 targets MDM2 and p21, in cancer cell lines with wild-type TP53 (SJSA-1, MOLM-13, DOHH-2, and WM-115), showing around 10-fold potent growth inhibition effects compared to Nutlin-3a (Table 1). The xenograft mouse models with SJSA-1 and MOLM-13 cells showed > 90% reduction in tumor growth with oral administrations of 25 and 50 mg/kg/day. For discovering predictive gene signatures, we performed two different approaches. In the first approach, 240 cell lines available as OncoPanel were treated with DS-3032b, another prototypic MDM2 inhibitor DS-5272, and Nutlin-3a, and determined 62 sensitive and 164 resistant lines, based on GI50s. Using gene expression profiling (GEP) publicly available for all the cell lines, we selected 175 top-ranked genes with highest expression in the 62 sensitive cell lines. We thus defined the average of Z-scores of the 175 gene expression as "sensitivity score". To validate the 175-gene signature, we evaluated in vivo anti-tumor activities of DS-3032b in 13 patient-derived tumor xenografts (melanoma, NSCLC, colorectal and pancreatic cancers). The prediction accuracy, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) were 85, 88, 88 and 80% respectively. As another validation set, 41 primary AML samples were treated with DS-3032b to define the top and bottom one-third most sensitive or resistant samples (14 each), and GEP was performed in every sample. TP53 mutations were detected in 8 specimens by next generation sequencing and confirmed by Sanger sequencing. The 175-gene signature was applied to the AML dataset, and the accuracy, sensitivity, PPV and NPV to predict the 14 sensitive or resistant samples were 79, 93, 72 and 90% respectively. Importantly, this signature was more predictive than the TP53 mutation status alone applied (68, 93, 62 and 86%). (Table 2A-B) In contrast to the cell line-based approach, the second approach defined an AML-specific gene signature. Specifically, we used the same dataset of 41 primary AML samples described above as training and validation set, by performing random forest methods with cross validation. Using a routine way in bioinformatics analysis of classifying gene signature, we first selected the 1500 top-ranked genes with highest expression variance among all the specimens. In addition, p53-related 32 genes that potentially have predictive values were also selected based on the previous reports. Classification was performed using the random forest method to identify a predictive algorithm with the 1500-gene set, 32-gene set or combined 1525-gene set (7 genes were overlapped), thus we found that the 1525-gene set had highest performance than each gene set alone. However, applying this method to all the 41 samples showed inferior predictive performance than applied only to the 33 wild-type TP53 samples (the prediction accuracy, sensitivity, PPV and NPV were 68, 72, 67 and 69%, vs. 77, 82, 75 and 80%).(Table 2C) Finally, we combined each of the two algorithms (Table 2B-C) with TP53 mutation status. Specifically, the samples with TP53 mutations were predicted as resistant, then either of gene signatures was applied to the rest of the samples with wild-type TP53. Predictive performance (Table 2D-E) was improved in both signatures compared to the others, especially showing the highest PPVs (80 and 82%, respectively). Taken together, gene signatures discovered in the present study, by combining with TP53 mutation status, provided new highly predictive algorithms for therapy of MDM2 inhibition. Our findings will be tested in ongoing clinical trials of DS-3032b. Disclosures Nakamaru: Daiichi Sankyo Co., Ltd: Employment. Seki:2Daiichi Sankyo Co., Ltd.: Employment. Tazaki:2Daiichi Sankyo Co., Ltd.: Employment. DiNardo:Celgene: Research Funding; Novartis: Other: advisory board, Research Funding; Abbvie: Research Funding; Agios: Other: advisory board, Research Funding; Daiichi Sankyo: Other: advisory board, Research Funding. Tse:Daiichi Sankyo, Inc.: Employment.

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Novel Small Molecule MDM2 Inhibitor MI-63 Induces p53-Dependent Apoptosis in AML Cell Lines.

Blood ◽

10.1182/blood.v108.11.2596.2596 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2596-2596

Author(s):

Ismael Samudio ◽

Martin Dietrich ◽

Paul Corn ◽

Dajun Yang ◽

Gautam Borthakur

Keyword(s):

Cell Line ◽

Cell Lines ◽

Small Molecule ◽

Negative Regulator ◽

Dependent Manner ◽

Wild Type ◽

Wild Type P53 ◽

Induced Apoptosis ◽

Related Proteins ◽

Mdm2 Inhibitor

Abstract Although TP53 mutations are rare in acute myeloid leukemia (AML), inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2 (murine double minute 2). We investigated the effects of MI-63, a small molecule that activates p53 by inhibition of MDM2-p53 interaction [ Ki value of 3 nM (J Med Chem.2006;49(12):3432–5)] in AML cell lines. Treatment with MI-63 triggered apoptosis (evidenced by loss of membrane potential and externalization of phosphatidylserine) in AML cell lines with wild-type p53 (OCI-AML-3 and MOLM13) in a time and concentration-dependent manner (IC50 at 72 hrs.= 2.5 μM for OCI-AML-3 and 1 μM for MOLM-13), while a p53-null AML cell line (HL-60) was resistant (IC50 not reached at 10 μM). Moreover, knockdown of p53 in OCI-AML3 cells rendered this cell line resistant to MI-63 induced apoptosis while control vector infected OCI-AML-3 cells remained as sensitive to MI-63 similar to the parental cells. Mechanistic studies showed that MI-63 blocks G1/S phase transition in AML cells with wild-type p53 resulting in accumulation of cells in G1 phase (percentage cells inG1 phase at 24 hrs. = 88.66% vs 43.49% in cultures with DMSO control) while MI-61, a skeletally related but inactive control compound failed to do so (41.63%). Treatment with MI-63 increased cellular levels of p53 and p53 dependent proteins in OCI-AML-3 cells that include p21 and BH3-only pro-apoptotic protein Puma and pro-apoptotic multi-domain Bcl-2 family member Bax. Additionally, MI-63 induced a profound decrease in the levels of MDM4, an MDM2 homolog that has been reported to mediate resistance to the effects of nutlin-3a, suggesting that MI-63 may offer a therapeutic advantage in cells expressing high levels of MDM4. Finally, supporting the concept that increased levels of p53 modulate the apoptotic rheostat both directly, by behaving as a BH3-only protein, and indirectly by increasing the levels of sensitizer BH3-only proteins, MI-63 potently synergized with AT-101, an orally available pan inhibitor of Bcl-2, Bcl-xL and Mcl-1 (currently being evaluated as an antitumor agent in Phase I/II trials by Ascenta Therapeutics), to induce mitochondrial dysfunction and apoptosis in OCI-AML-3 cells (average combination index = 0.055±0.019). Taken together our results support preclinical evaluation of novel small molecule MI-63 alone and in combination with Bcl-2 inhibitors for the therapy of AML. The studies in primary AML samples are ongoing. Fig.1: MI-63 Induced Apoptosis Requires Intact p53 Fig.1:. MI-63 Induced Apoptosis Requires Intact p53 Fig.2: Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included) Fig.2:. Efect of MI-63 on p53 and Related Proteins (comparison with N3a, a known MDM2 inhibitor included)

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Role Of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) and Its Ligands In The Regulation Of Functional OCT-1 Activity In CML Cells

Blood ◽

10.1182/blood.v122.21.1470.1470 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1470-1470

Author(s):

Jueqiong Wang ◽

Chung Hoow Kok ◽

Richard J. D'Andrea ◽

Timothy P. Hughes ◽

Deborah L. White

Keyword(s):

Cell Death ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

K562 Cells ◽

Vehicle Control ◽

Peroxisome Proliferator ◽

Advisory Committees ◽

Peroxisome Proliferator Activated Receptor

Abstract Introduction The human organic cation transporter-1 (hOCT-1) is the primary active influx protein for imatinib in BCR-ABL positive cells. The functional activity of the OCT-1 protein (OCT-1 activity, OA) is predictive of molecular response in de-novo chronic phase chronic myeloid leukemia (CP-CML) patients. We have previously demonstrated that diclofenac, a competitive peroxisome proliferator-activated receptor-γ (PPARγ) antagonist, can significantly increase OA in CML cells 1. However, the role of PPARγ and its ligands in OA regulation remain unknown. Thus, the link between OA and PPARγ in CML cells has been investigated in this study. Methods OA was determined by intracellular uptake and retention assay (IUR) in the presence and absence of the OCT-1 inhibitor, prazosin 2. To assess the effect of PPARγ ligands on OA, BCR-ABL positive cell lines (KU812, K562) were incubated with PPARγ antagonist (GW9662, T0070907) or agonists (GW1929, rosiglitazone) respectively for 1 hour immediately prior to the IUR assays. The OA was also assessed in the mononuclear cells (MNCs) of 77 CP-CML patients enrolled to the TIDEL II trial. PPARγ activity in CML MNC nuclear extracts was determined through the use of a PPARγ Transcription Factor Assay Kits according to the manufacturer's instructions. To assess the effect of PPARγ ligands on cell death, KU812 or K562 cells were stained with AnnexinV and 7-AAD for detection of apoptosis after the co-administration of imatinib and PPARγ ligands for 72 hours. Results A significant increase in OA was observed in KU812 and K562 cells treated with PPARγ antagonists. In contrast, PPARγ agonists significantly decreased the OA in both cell lines (Table 1). A negative link between OA and PPARγ activity was observed in CML MNC samples (R=-0.585, p<0.001). PPARγ activity was significantly elevated in CML patients who had a low OA at diagnosis (less than 4 ng/200,000 cells) compared with those who had higher OA (p<0.001). After 72 hours co-administration with 0.1µM imatinib, KU812 cells treated with PPARγ antagonists (GW9662 and T0070907) showed a significantly lower cell viability (40% and 18% respectively) compared with vehicle control (70%, p<0.001). Similar results were also observed in K562 cells after co-administration with 1.0µM imatinib for 72 hours. K562 cells treated with PPARγ antagonists (GW9662: 51% and T0070907: 47%) showed a significantly lower cell viability (51% and 47% respectively) compared with vehicle control (61%, p<0.05). Conclusion Ligand-activation or inhibition of PPARγ is a regulator of OA in CML cell lines, and the low MNC OCT-1 activity in CML patients is consistent with the high level of PPARγ activity in these cells. Low PPARγ activity may be the key driver for low OA and poor imatinib response observed in a subset of CML patients. Importantly, the enhanced OA as a result of PPARγ antagonist treatment resulted in increased cell death following co-administration with imatinib. Ongoing studies relating to the upstream pathways involved in PPARγ activation aim to reveal the possible mechanism of OA modulation by PPARγ. Enhancement of OA by PPARg antagonists is likely to provide an important axis for clinical application to improve the clinical efficacy of imatinib. This would be particularly important in patients with low OA who currently have inferior outcomes with imatinib therapy. 1. Wang J, Hughes TP, Kok CH, et al. Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells. British Journal of Cancer. 2012;106(11):1772-1778. 2. White DL, Saunders VA, Dang P, et al. Most CML patients who have a suboptimal response to imatinib have low OCT-1 activity: Higher doses of imatinib may overcome the negative impact of low OCT-1 activity. Blood. 2007;110(12):4064-4072. Disclosures: Hughes: Novartis: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

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Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinases Is Associated with Multiple Myeloma Progression, Prognosis and Extramedullary Plasmacytoma

Blood ◽

10.1182/blood-2018-99-113355 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3178-3178

Author(s):

Rei Ishihara ◽

Yuki Murakami ◽

Kazuki Homma ◽

Saki Watanabe ◽

Tsukasa Oda ◽

...

Keyword(s):

Multiple Myeloma ◽

Matrix Metalloproteinase ◽

Cell Lines ◽

Research Funding ◽

Poor Prognosis ◽

Extramedullary Plasmacytoma ◽

Negative Regulator ◽

Expression Level ◽

Tissue Inhibitor Of Metalloproteinases ◽

Tissue Inhibitor

Abstract Backgrounds and Aims: Matrix metalloproteinase (MMP) is endopeptidase enzyme degrading extracellular matrix, and tissue inhibitor of metalloproteinases (TIMP) is negative regulator of MMP. MMP is well known to be involved in metastatic mechanism of cancer cell and oncogenesis. However expression and role of MMP and TIMP has not been well established in multiple myeloma (MM). Therefore we examined whether expression of MMP and TIMP was involved in progression and prognosis of MM and extramedullary plasmacytoma (EMP) formation. Materials and Methods: Purified bone marrow plasma cells by using anti-CD138 antibody and magnetic beads obtained from 151 MM, 64 MGUS, 18 control and 5 EMP were subjected to the study after informed consent. The study was approved by IRB following Declaration of Helsinki. Whole transcriptome by next generation sequencer (NGS) using Illumina Next Seq 500 was performed in part of the samples to select genes to be studied, then expression level of MMP and TIMP determined by RQ-PCR Delta Ct value normalized with ACTB was used for analysis. MM cell lines KMS11, KMS12PE, KMS12BM, KMM1, RPMI8226, MM1S were used for the in vitro study. Results: We selected TIMP-1, 2and MMP14, 24 based on transcriptome analysis data comparing MM and EMP. The expression level of TIMP1 and MMP24 was significantly higher in MM (median delta Ct: 0.033 for TIMP1, 0.00025 for MMP24) than in MGUS (median delta Ct: 0.013 for TIMP1, 0.00006 for MMP24) (p=0.005, p=0.001), however TIMP2, MMP14 level did not differ in between MM and MGUS. Interestingly, TIMP1, 2 and MMP14, MMP24 were expressed with strikingly higher levels in EMP than in MM with 20 times, 60 times, 300 times, 500 times respectively (p=0.01, p=0.02, p=0.004, p=0.004). Both TIMP1 and 2 expression were higher in the MM patients with high risk cytogenetic karyotype t(4;14), t(14;16), del 17p than in the patients without such karyotype (p=0.006, p=0.008), however the levels of MMP14 and 24 did not differ in between cytogenetic risk groups. Positive correlations were found in between TIMP1 and 2, MMP14 and 24 in both MM and MGUS group respectively (r=0.34, p<0.001, r=0.49, p<0.001 in MM, r=0.32, p=0.008, r=0.63, p<0.001 in MGUS). Since TIMP2 and MMP14 were higher in EMP, the effect of recombinant TIMP2, siRNA-TIMP2 and MMP14 inhibitors marimastat/ilomastat were tested in MM cell lines KMM1, KMS11, KMS12PE and RPMI8226. However, these interventions did not change proliferation rates of these cell lines. The cell lines were treated with doxorubicin or bortezomib to study if these agent can change TIMP and MMP expression. Doxorubicin significantly increased expression level of MMP14, 24 and TIMP1, 2, but bortezomib increased only MMP14, 24. In the newly diagnosed MM (NDMM) patients (n=77), median time of overall survival (OS) of the patients with high TIMP1 (more than median value) was significantly shorter (2.7 years vs not reached) and 3 year OS rate was inferior (40% vs 65%) (p=0.0095). Progression free survival (PFS) tended to be inferior for high TIMP1 group, but the difference did not reach statistical significance (p=0.221). OS and PFS were not different according to either TIMP2, MMP14 or MMP24. Conclusions: Our results suggest that TIMP1, 2 and MMP14, 24 were associated with EMP formation. Among those factors, TIMP1 is the one which may play a key role for MM progression and chemo-resistance based on the results revealing its upregulation by antineoplastic agents and association with poor prognosis of MM patients. Our results is consistent with a previous report describing that high serum TIMP1 concentration was associated with poor prognosis of MM. TIMP is recently shown to play another role besides negative regulator for MMP, so further study to elucidate its specific role for chemo-resistance contributes to develop novel therapy targeting TIMP and MMP pathway. Disclosures Tsukamoto: Kyowa-Kirin: Research Funding; Chugai: Research Funding; Eisai: Research Funding; Pfizer: Research Funding. Handa:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.

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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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Activation of p53 By Novel MDM2 Antagonist RG7388 Overcomes AML Inherent and Acquired Resistance to Bcl-2 Inhibitor ABT-199 (GDC-0199)

Blood ◽

10.1182/blood.v124.21.2162.2162 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2162-2162 ◽

Cited By ~ 1

Author(s):

Rongqing Pan ◽

Kensuke Kojima ◽

Zhuanzhen Zheng ◽

Vivian R Ruvolo ◽

Gwen Nichols ◽

...

Keyword(s):

Cell Lines ◽

Research Funding ◽

Acquired Resistance ◽

Model Systems ◽

Employment Equity ◽

P53 Activation ◽

Equity Ownership ◽

Induced Apoptosis ◽

Mdm2 Overexpression ◽

Mdm2 Inhibitor

Abstract Background: Acute myeloid leukemia (AML) is characterized by the clonal expansion of immature myeloid cells. AML is treated primarily with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel therapies to achieve higher cure rates with more acceptable toxicities. Bcl-2 family proteins, together with TP53, are the central regulators of apoptosis. Overexpression of Bcl-2 protein is associated with leukemia progression and chemoresistance. We have observed elevated expression of Bcl-2 in AML and recently demonstrated that Bcl-2 inhibition by ABT-199 effectively induced apoptosis in AML (Pan, et.al., Cancer Discovery, 2014). However, resistance to ABT-199 was observed in cells expressing high levels of Mcl-1 or Bcl-xL. Moreover, a recent study showed heterogeneous but overlapping expression of Bcl-2, Mcl-1, and Bcl-xL proteins in 577 AML patient samples (Bogenberger, et. al., Leukemia, 2014). Although common in solid tumors, p53 mutations are relatively rare in AML. However, p53 functions are diminished by overexpression of MDM2 protein, an E3 ubiquitin ligase of p53 and an inhibitor of p53 transactivation. We previously reported MDM2 overexpression in 53% of primary AMLs (Kojima et al., Blood, 2005). Our group also demonstrated that p53 activation by Nutlins, the prototypical MDM2 inhibitors, induced apoptosis and growth inhibition in AML. Rationale: Since p53 activation by MDM2 inhibitors upregulates pro-apoptotic Bcl-2 proteins like NOXA, PUMA, and Bax, which counteract Mcl-1 and Bcl-xL, we hypothesized that the second-generation MDM2 inhibitor RG7388 could overcome AML resistance to Bcl-2-specific ABT-199, and that the combination could synergistically enhance apoptosis in AML. Results: We first demonstrated that RG7388 induced apoptosis exclusively in p53 wild type (wt) cells. RG7388 was essentially ineffective in p53 mutant or null AML cell lines such as HL-60, KG1 and THP1 (48h IC50s > 5 μM). Nonetheless, it showed high potency against p53 wt cell lines (48h IC50s: MOLM13 = 21.7 nM, MV-4-11 = 29.2 nM). Furthermore, stable knockdown of TP53 rendered the wt cell lines completely resistant to RG7388 (IC50s > 5 μM), confirming TP53-specificity. To study if RG7388 was able to overcome inherent resistance to ABT-199, we tested its efficacy on OCI-AML3 cells, which are inherently resistant to ABT-199, AraC and Idarubicin. As a single agent, RG7388 potently killed OCI-AML3 cells (48h IC50 = 148 nM). Importantly, RG7388 was ~20-fold more effective in OCI-AML3 cells than its predecessors Nutlin-3a and RG7112. We also examined the time- and dose-response of RG7388 in several genetically diverse AML cell lines (p53 wt) and found that 100 nM RG7388 was able to induce apoptosis and inhibit cell growth within 12 h. Next we studied whether RG7388 synergizes with ABT-199 to kill the refractory OCI-AML3 cells. A combination index of 0.35 (Chou-Talalay method) indicated a strong synergy between the two compounds. The combination exhibited higher activity in killing OCI-AML3 cells than either agent alone (48h IC50s: ABT-199 = 1680 nM, RG7388 = 148 nM, ABT+RG = 28 nM). Similar synergy was observed in additional AML cell lines and in primary samples. Next, we generated ABT-199 resistant cells by continuous exposure of initially sensitive AML cells to escalating concentrations of ABT-199. While 1000 nM ABT-199 had no effects on the viability of these cells, additional treatment with 30 nM RG7388 effectively killed them. This finding suggested that RG7388 was able to overcome acquired resistance to ABT-199. The mechanisms underlying this resensitization and its synergism with ABT-199 are under investigation using in vitro and in vivo model systems. Conclusions: The novel MDM2 inhibitor RG7388 induces growth arrest and apoptosis selectively in p53 wt AML cells. Importantly, the combination of RG7388 with ABT-199 synergistically induced apoptosis in AML cell lines and primary patient cells, and RG7388 was able to overcome inherent or acquired resistance to ABT-199. Since both Bcl-2 and MDM2 overexpression are associated with poor prognosis in AML, the proposed combination of the two clinical-stage compounds could have considerable clinical potential. We will report on ongoing experiments with primary AML cells in NSG mice to determine the potential of this combinatorial approach to eliminate AML stem cells. Disclosures Nichols: Roche: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership. Dangl:Roche: Employment, Patents & Royalties. Konopleva:Abbvie: Research Funding. Andreeff:Roche: Research Funding; Abbvie: Research Funding.

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The Chromosome 21 Kinase DYRK1A and Its Substrate FOXO1 Constitute a Novel Therapeutic Pathway in B-ALL

Blood ◽

10.1182/blood-2018-99-119854 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 548-548

Author(s):

Rahul S. Bhansali ◽

Malini Rammohan ◽

Ji Heon Paul Lee ◽

Sebastien Malinge ◽

Yi-Chien Tsai ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

B Cells ◽

Cell Lines ◽

Research Funding ◽

Negative Regulator ◽

Chromosome 21 ◽

Cyclin D3 ◽

B Lymphopoiesis

Abstract Dual Specificity Tyrosine-Phosphorylation-Regulated Kinase 1A (DYRK1A) is a serine/threonine kinase that regulates diverse pathways such as splicing, cell cycle, differentiation, apoptosis, and transcription. DYRK1A is encoded within the Down syndrome (DS) critical region of chromosome 21, underlying its importance in DS-related pathologies, such as Alzheimer's disease. Children with DS have an increased risk of developing hematologic malignancies, namely acute megakaryoblastic leukemia (DS-AMKL) and B-cell acute lymphoblastic leukemia (DS-ALL). We previously reported that DYRK1A promotes DS-AMKL by regulating subcellular localization of its substrate NFAT. In a subsequent study, we examined its role in normal hematopoiesis and found that DYRK1A is necessary for B and T cell development through phosphorylation and destabilization of Cyclin D3. Dyrk1a-deficient large pre-B cells and double negative thymocytes are unable to enter quiescence for maturation. Despite elevated levels of Cyclin D3, however, these cells lose proliferative capacity due to a block at the G2-M transition. This observation suggests that DYRK1A inhibition may exhibit anti-tumor activity in lymphocytes by first stimulating exit from quiescence but then blocking repeated rounds of cell division. Notably, DYRK1A is overexpressed in acute leukemias, including both T-ALL and B-ALL, relative to normal hematopoietic counterparts. Moreover, overexpression of dominant-negative DYRK1A-K188R impairs proliferation in human B-ALL cell lines, suggesting that DYRK1A kinase activity is required for B-ALL growth. In order to assess the physiologic relevance of targeting DYRK1A in vivo, we generated a murine model of B-ALL with a floxed Dyrk1a allele and observed significant survival advantages with homozygous (p=0.0045) and heterozygous deletion (p=0.0015). Additionally, both B-ALL cell lines and patient samples were sensitive to EHT1610, a potent and selective DYRK1 inhibitor. Relevant to the localization of DYRK1A on chromosome 21, DS-ALL samples were especially sensitive to kinase inhibition. EHT1610 also conferred synergistic growth inhibition of B-ALL cells when combined with cytotoxic chemotherapy drugs used in traditional ALL treatment regimens, such as dexamethasone, methotrexate and cytarabine. We next aimed to elucidate the mechanism by which DYRK1A inhibition cause a failure of G2-M progression. Using global and directed phosphoproteomic studies, we identified several DYRK1A substrates in pre-B cells that are involved in cell cycle, splicing, transcriptional regulation, and RNA metabolism. In addition to Cyclin D3, a notable substrate is FOXO1, an indispensable transcription factor in B lymphopoiesis. We observed that inhibition of DYRK1A led to an accumulation of FOXO1 in the nucleus of large pre-B cells despite intact PI3K/Akt signaling, which is the predominant negative regulator of FOXO1. Treatment of pre-B cells with AS1842856, an inhibitor of FOXO1 nuclear translocation, rescued the G2-M blockade and proliferative impairment induced by EHT1610 treatment. Despite FOXO1 acting as a tumor suppressor in normal lymphocytes, B-ALL cell lines and patient samples were paradoxically sensitive to FOXO1 inhibition, suggesting a unique requirement in the survival of B-ALL cells. This may be due to regulation of DNA damage, as DYRK1A inhibition alone led to negligible changes in gamma-H2AX foci, whereas FOXO1 inhibition increased DNA damage. When DYRK1A and FOXO1 were inhibited in combination, we observed a synergistic accumulation of DNA damage along with cell death in B-ALL cell lines. Finally, as both EHT1610 and AS1842856 are potent inhibitors of B-ALL cell growth in vitro, we assessed their in vivo efficacy. Both EHT1610 and AS1842856 significantly increased survival in xenograft models of B-ALL (p=0.0002 and p=0.001, respectively). We therefore conclude that both DYRK1A and its substrate FOXO1 are therapeutic targets in B-ALL. Importantly, EHT1610 represents the first selective DYRK1A inhibitor with suitable in vivo activity. Ultimately, we have determined that the DYRK1A pathway is integral to the maintenance of normal and malignant B-lymphopoiesis, the latter which can be effectively targeted through 1) a primary proliferative impairment, 2) sensitization to cell cycle-dependent chemotherapy, and 3) downstream inhibition of DYRK1A substrates such as FOXO1. Disclosures Lee: AbbVie: Employment. Bourquin:Amgen: Other: Travel Support. Crispino:Scholar Rock: Research Funding; Forma Therapeutics: Research Funding.

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Targeting DDX3X Helicase Activity with BA103 Shows Promising Therapeutic Effects in Preclinical Glioblastoma Models

Cancers ◽

10.3390/cancers13215569 ◽

2021 ◽

Vol 13 (21) ◽

pp. 5569

Author(s):

Annalaura Brai ◽

Valentina Riva ◽

Letizia Clementi ◽

Lucia Falsitta ◽

Claudio Zamperini ◽

...

Keyword(s):

Cell Lines ◽

Antitumoral Activity ◽

Therapeutic Effects ◽

Helicase Activity ◽

In Vivo Evaluation ◽

Cancer Types ◽

And Migration ◽

Oncogenic Protein ◽

New Compounds

DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.

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