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

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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BCL-2 Inhibition By ABT-199 (Venetoclax/GDC-0199) and p53 Activation By RG7388 (Idasanutlin) Reciprocally Overcome Leukemia Apoptosis Resistance to Either Strategy Alone: Efficacy and Mechanisms

Blood ◽

10.1182/blood.v126.23.673.673 ◽

2015 ◽

Vol 126 (23) ◽

pp. 673-673 ◽

Cited By ~ 2

Author(s):

Rongqing Pan ◽

Vivian Ruvolo ◽

Hong Mu ◽

Zhuanzhen Zheng ◽

Joel Leverson ◽

...

Keyword(s):

Research Funding ◽

Acquired Resistance ◽

Cell Cycle Distribution ◽

G1 Arrest ◽

Apoptosis Resistance ◽

Employment Equity ◽

P53 Activation ◽

Equity Ownership ◽

Mdm2 Inhibitor ◽

Refractory Aml

Abstract Acute myeloid leukemia (AML) is primarily treated with chemotherapy, but the 5-year survival rate has only marginally increased over the past few decades, highlighting the need for novel targeted therapy. We have reported elevated expression of BCL-2 in AML and that BCL-2 inhibition by ABT-199 (ABT, venetoclax) induced on-target apoptosis, which could be predicted by BH3 profiling (Pan, et al., Cancer Discovery, 2014). ABT also showed encouraging clinical activity in relapsed/refractory AML (Konopleva et al., ASH 2014), yet MCL-1-mediated resistance may limit its use as monotherapy. p53 mutations are relatively rare in AML, but its functions are often suppressed by overexpressed MDM2 protein. Since p53 and BCL-2 family proteins are central regulators of apoptosis, we asked whether concurrent BCL-2 inhibition and p53 activation (by MDM2 inhibitor) could overcome resistance to apoptosis and synergistically induce apoptosis in AML cells. The novel MDM2 inhibitor RG7388 (RG, Idasanutlin) robustly activated p53 and induced growth inhibition and apoptosis of AML cells in a p53-dependent manner. p53 activation by RG also synergized with BCL-2 inhibition in killing ABT-sensitive cell lines such as MOLM-13 or MV-4-11 (Fig. 1A). After long-term exposure to escalating doses of ABT, initially sensitive cells upregulated MCL-1 and acquired resistance. Nonetheless, the acquired resistance could be effectively abrogated by RG (Fig. 1B). OCI-AML3 cells express a high basal level of MCL-1, and are inherently resistant to ABT. Concurrent p53 activation and BCL-2 inhibition induced synergistic apoptosis and overcame the inherent ABT resistance (Fig. 1C). Next, we studied the underlying mechanisms. p53 activation by RG increased the expression of PUMA and BAX (but not NOXA, Fig. 1D), which are able to counteract MCL-1. In addition, p53 activation quickly dephosphorylated ERK2 and downregulated MCL-1 (Fig. 1E). Surprisingly, ABT increased ERK2 phosphorylation and upregulated MCL-1 (Fig. 1E). Because active ERK2 phosphorylates and stabilizes MCL-1, these results indicate that the observed changes in MCL-1 levels could be attributed to ERK2 phosphorylation status. Consistently, ERK2 dephosphorylation by MEK inhibitors quickly reduced MCL-1. Most importantly, ABT-induced ERK2 phosphorylation and MCL-1 upregulation could be reversed by p53 activation (Fig. 1F). These mechanistic studies provide insights into how p53 activation overcomes acquired/inherent resistance to BCL-2 inhibition. OCI-AML3 cells are relatively resistant to p53 activation by RG. We used concomitant Annexin V staining, EdU pulsing and PI staining to simultaneously analyze apoptosis induction and cell cycle distribution of live cells (Fig. 1G). p53 activation by RG induced cell accumulation in G1 phase, while little apoptosis occurred (Fig. 1H). Addition of ABT dramatically increased apoptosis, reduced G1-arrested cells, and boosted apoptotic hallmarks like the cleavage of caspase-9, -3 and PARP-1 (Fig. 1H-I). ABT did not affect p21 expression and cell cycle distribution, and p53 activation induced robust expression of p21 and G1 arrest. Furthermore, p21 knockdown significantly decreased G1-arrested cells and increased apoptosis following p53 activation, indicating that p21 upregulation and G1 arrest mediate apoptosis resistance to p53 activation. Nonetheless, addition of ABT effectively shifted cell response from G1 arrest to apoptosis, suggesting BCL-2 inhibition can reciprocally overcome apoptosis resistance to p53 activation. Next, we tested the combination in two AML mouse models. In an OCI-AML3-derived mouse model (with inherent resistance to ABT or RG), ABT or RG prolonged survival by 10 or 19 d, respectively, while the combination prolonged mouse survival by 61 d (Fig. 1J-K). Currently, we are also following the survival of mice in a MOLM-13 acquired resistance model. Early results indicate the tumor burden in combination group is <1/100 of that in control/ABT groups and ~1/20 of that in the RG group at day 14 (Fig. 1K-L). In summary, BCL-2 inhibition by ABT and p53 activation by RG can reciprocally overcome resistance to apoptosis encountered by using either treatment alone in vitro and in vivo. 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 impact in relapsed/refractory AML. Disclosures Leverson: AbbVie: Employment, Equity Ownership. Konopleva:Novartis: Research Funding; AbbVie: Research Funding; Stemline: Research Funding; Calithera: Research Funding; Threshold: Research Funding. Nichols:Roche Pharma: Employment, Equity Ownership.

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CC-92480 Is a Novel Cereblon E3 Ligase Modulator with Enhanced Tumoricidal and Immunomodulatory Activity Against Sensitive and Resistant Multiple Myeloma Cells

Blood ◽

10.1182/blood-2019-124338 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1812-1812 ◽

Cited By ~ 3

Author(s):

Antonia Lopez-Girona ◽

Courtney G. Havens ◽

Gang Lu ◽

Emily Rychak ◽

Derek Mendy ◽

...

Keyword(s):

Cell Lines ◽

Acquired Resistance ◽

E3 Ligase ◽

Immunomodulatory Activity ◽

Employment Equity ◽

Ic50 Value ◽

Ic50 Values ◽

Equity Ownership ◽

Induction Of Apoptosis ◽

Low Levels

Lenalidomide- and pomalidomide-based therapies are effective drugs in the treatment of patients with multiple myeloma (MM), however most patients with MM eventually relapse or become resistant. CC-92480, a novel cereblon (CRBN) E3 ligase modulator (CELMoD) with multiple activities including potent immunomodulation and single-agent antiproliferative effects, is being investigated in a phase 1 clinical trial (CC-92480-MM-001; NCT03374085) for patients with relapsed/refractory MM (RRMM). The present study investigates the preclinical data and mechanism of action of CC-92480 in MM models. CELMoD agents bound to CRBN confer differentiated substrate-degradation specificity on the CRL4CRBN E3 ubiquitin ligase. CRBN-modulator agents mediate destruction of Ikaros and Aiolos, transcription factors that contribute to myeloma cell survival. CC-92480 was found to produce rapid, deep, and sustained degradation of Ikaros and Aiolos, with superior antimyeloma activity. Accordingly, in a CRBN protein competitive binding assay, CC-92480 displaced a Cy-5-labeled CELMoD analog from CRBN with a 50% inhibitory concentration (IC50) value of 0.03 μM, whereas lenalidomide competed with an IC50 value of 1.27 μM in the same assay, demonstrating a higher binding affinity of CC-92480 for CRBN. Additionally, CC-92480 promoted the recruitment of Ikaros to the CRBN E3 ligase complex more effectively than pomalidomide in 2 orthogonal CRBN/Ikaros binding assays; it also triggered a more extensive cellular ubiquitination of Ikaros, and a faster, more efficient depletion of cellular Ikaros and Aiolos than pomalidomide. In various MM cell lines, including those with acquired resistance to lenalidomide or pomalidomide and low levels of CRBN, CC-92480 produced robust degradation of Ikaros and Aiolos followed by strong reduction of 2 additional and highly critical transcription factors, c-Myc and interferon regulatory factor 4, which are linked to the induction of apoptosis as measured by cleaved caspase-3. The tumoricidal activity of CC-92480 was shown to be CRBN dependent, since the effect was prevented by complete loss of CRBN or by the stabilization of Ikaros and Aiolos. CC-92480 displayed broad and potent antiproliferative activity across a panel of 20 MM cell lines that are either sensitive, have acquired resistance, or are refractory to lenalidomide or pomalidomide; the cell lines also contained diverse chromosomal translocations and oncogenic drivers typically found in MM patients. Approximately half of the MM cell lines evaluated were highly sensitive to CC-92480, with IC50 values for antiproliferative activity ranging from 0.04 to 5 nM; only 2 cell lines had IC50 values > 100 nM. CC-92480 inhibits cell proliferation and induces apoptosis in MM cell lines that are not sensitive to lenalidomide or pomalidomide. This panel of cell lines includes both refractory cell lines and resistant cell lines generated through continuous exposure to lenalidomide and pomalidomide that acquired low levels of CRBN protein or mutations in the CRBN gene. CC-92480 also induced deep destruction of Ikaros and Aiolos in cultures of peripheral blood mononuclear cells (PBMCs), which led to the activation of T cells and increased production of the cytokines interleukin-2 and interferon gamma. These responses occurred at the range of CC-92480 concentrations that show potent tumoricidal effect against MM cells. The T cell activation and enhanced cytokine production by CC-92480 led to the potent and effective immune-mediated killing of MM cells in co-cultures with PBMCs. CC-92480 is a potent antiproliferative and proapoptotic novel CELMoD with enhanced autonomous cell-killing activity in MM cells that are either sensitive, resistant, or have acquired resistance to lenalidomide and pomalidomide. CC-92480 has a unique and rapid degradation profile stemming from the enhanced efficiency to drive the formation of a protein-protein interaction between Ikaros and Aiolos and CRBN, inducing cytotoxic effects in a CRL4CRBN-dependent fashion that leads ultimately to the induction of apoptosis, even in the context of low or mutated CRBN protein. Additionally, similar to lenalidomide, CC-92480 conserves immunomodulatory activity against MM cells. These data support the clinical investigation of CC-92480 in patients with RRMM. Disclosures Lopez-Girona: Celgene Corporation: Employment. Havens:Pfizer: Employment, Equity Ownership; Celgene: Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Rychak:Celgene Corporation: Employment, Equity Ownership. Mendy:Celgene Corporation: Employment. Gaffney:Celgene: Employment. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Matyskiela:Celgene corporation: Employment. Khambatta:Celgene: Employment. Wong:Celgene Corporation: Employment, Equity Ownership. Hansen:Celgene Corporation: Employment. Pierce:Celgene Corporation: Employment, Equity Ownership. Cathers:Global Blood Therapeutics (GBT): Employment; Celgene Corporation: Equity Ownership. Carmichael:Celgene plc: Employment, Equity Ownership.

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Absence Of Mutation In Cereblon (CRBN) and DNA Damage Binding Protein 1 (DDB1) Genes In Myeloma Cells and Patients and Its Clinical Significance

Blood ◽

10.1182/blood.v122.21.3139.3139 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3139-3139

Author(s):

Anjan Thakurta ◽

Anita K Gandhi ◽

Michelle Waldman ◽

Chad C. Bjorklund ◽

Suzanne Lentzsch ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Heterozygous Mutation ◽

Employment Equity ◽

Advisory Committees ◽

Single Nucleotide ◽

Truncating Mutation ◽

Equity Ownership

Abstract Background CRBN, a target of thalidomide and IMiDs® immunomodulatory agents lenalidomide (LEN) and pomalidomide (POM), is a component of the E3 ubiquitin cullin 4 ring ligase (CRL4) complex that also includes DDB1, Roc1, and Cul4. Two CRBN mutations have been reported in multiple myeloma (MM) patients: truncating mutation (Q99) and point mutation (R283K). One copy of the CRBN gene was shown to be deleted in the MM1S and MM1S.R cell lines. No DDB1 mutation has been described previously. Results We investigated the incidence of CRBN and DDB1 mutations by next-generation sequencing in 20 MM cell lines and MM subjects. Of 90 MM patients, 24 were newly diagnosed and 66 were relapsed and refractory of which 36 patients were LEN resistant. Out of the cell lines tested, 1 heterozygous CRBN mutation (D249Y) was found in the LEN-resistant ANBL6R cells, which is located in the putative DDB1 binding domain, and 2 single silent mutations were identified in the KMS-12-BM (rs17027638) and OPM-2 cells. One DDB1 heterozygous mutation (E303D) was identified in ANBL6 cells. In the cohort of patients assessed, no CRBN mutation was detected; however, 5 single nucleotide variations (SNV) were identified. Three of the 5 SNVs were at position 735 (Y245Y) and 1 each at position 219 (H73H) and 939 (C313C), respectively. The first 2 SNVs (rs17027638 and rs1045309) are described but not the last. We found a single SNV (P51P; rs2230356) in DDB1 gene the patient samples. Conclusion Mutations within the coding sequences of CRBN and DDB1 are rare in MM patients and cell lines. Most intrinsically LEN-resistant cells and cell lines made resistant to LEN or POM do not have CRBN or DDB1 mutations, suggesting the potential role of other sources, such as genetic or epigenetic pathways in developing resistance to IMiD drug–based therapy. Disclosures: Thakurta: Celgene: Employment, Equity Ownership. Gandhi:Celgene: Employment, Equity Ownership. Waldman:Celgene: Employment, Equity Ownership. Bjorklund:Celgene: Employment, Equity Ownership. Lentzsch:Celgene: Research Funding. Schey:Celgene: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; NAPP: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees. Madan:Covance Genomics Lab: Employment. Ning:Celgene: Employment, Equity Ownership. Mendy:Celgene: Employment, Equity Ownership. Lopez-Girona:Celgene: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Avet-Loiseau:Celgene: Research Funding. Chopra:Celgene: Employment, Equity Ownership.

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Degradation of Aiolos and Ikaros Followed By Downregulation of c-Myc and then IRF4 Leads to Programmed Cell Death in Myeloma Cells Sensitive to IMiDs® Immunomodulatory Compounds

Blood ◽

10.1182/blood.v124.21.4700.4700 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4700-4700

Author(s):

Chad C Bjorklund ◽

Courtney G. Havens ◽

Patrick R Hagner ◽

Anita K. Gandhi ◽

Maria Wang ◽

...

Keyword(s):

Cell Death ◽

Programmed Cell Death ◽

Protein Expression ◽

Cell Lines ◽

Acquired Resistance ◽

Clinical Samples ◽

Employment Equity ◽

Molecular Sequence ◽

Sequence Of Events ◽

Equity Ownership

Abstract Background: The zinc finger transcription factors, Aiolos (IKZF3) and Ikaros (IKZF1) were identified as lenalidomide (LEN) and pomalidomide (POM)-induced substrates of the cereblon (CRBN)-dependent Culin4 E3-ligase complex. While recent studies suggest that the anti-proliferative activity of LEN and POM in multiple myeloma (MM) cell lines in vitro is due in part to the targeted ubiquitination and subsequent proteasomal degradation of Aiolos and Ikaros, the downstream molecular mechanisms remain unknown. Using inducible shRNA-mediated knockdown combined with kinetic analyses, we systematically investigated the biological mechanisms associated with the degradation of Ikaros and Aiolos in MM cell lines that are sensitive to or have acquired resistance to LEN and POM. Results: In MM1.S and U266 MM cell lines stably engineered with doxycycline (DOXY)-inducible shRNAs, knockdown of either Ikaros or Aiolos showed a reduction in cell proliferation (80%-90%) as measured by 3H-thymidine incorporation after a 4 day treatment with DOXY. We demonstrated that this anti-proliferative effect is inherently tied to and precedes the induction of apoptosis, which was maximized (60%-80% AnnV+/ToPro3+) 5 days following Aiolos or Ikaros knockdown compared with a control shRNA. shRNA-mediated knockdown of Aiolos or Ikaros was furthermore associated with decreases in both c-Myc and IRF4 protein expression levels (70%-90% and 60%-80%, respectively) that were maximized by day 4. In turn, shRNA knockdown of either c-Myc or IRF4 elicited anti-proliferative (> 80% inhibition) and pro-apoptotic (50%-80%) responses as early as 48hrs after shRNA induction. These data suggest that the reduction of c-Myc and IRF4 protein levels downstream of Aiolos and Ikaros degradation account for the apoptotic effect and marks the onset of the cytotoxic response induced by LEN and POM in MM cells. To define the temporal order of events involving Aiolos, Ikaros, c-Myc and IRF4 in more detail, kinetic experiments following shRNA-mediated knockdown in parallel with drug treatments were performed. Data from these experiments showed that there is a distinct kinetic order of both LEN- and POM-mediated effects, initiated by immediate targeted degradation of Aiolos and Ikaros (within 90 min), followed by a decrease in c-Myc levels (24-48 hrs) with subsequent IRF4 downregulation (48-72 hrs), and finally, resulting in programmed cell death (3-5 days). Importantly, DOXY washout experiments, resulting in re-accumulation of Aiolos or Ikaros at early time points (24 hrs) partially overcame the antiproliferative effects of the shRNA-mediated knockdown of either target. Interestingly, upon the onset of c-Myc downregulation (24-48 hrs), the commitment to cell death could no longer be reversed in our experiments. Further, we generated MM1.S and U266 cells with acquired resistance to POM (10 µM; also cross-resistant to LEN) (MM1.S/PomR and U266/PomR , respectively), in which CRBN protein expression is substantially decreased (> 90%). Consequently, in these resistant cell lines, neither Aiolos nor Ikaros are degraded in the presence of LEN or POM. However, bypass of CRBN-dependent Aiolos degradation by DOXY-induced knockdown rescued c-Myc and IRF4 downregulation and concomitant inhibition of growth (90% and 60%, respectively), suggesting that resistant MM cells with acquired CRBN loss remain dependent on Aiolos and Ikaros. Conclusions: For the first time, our studies showed that degradation of Aiolos and Ikaros sets up a molecular sequence of events culminating in programmed cell death in MM cells. Our mechanistic studies showed that c-Myc is a key intermediate factor whose downregulation is a rate-limiting step for the transcriptional downregulation of IRF4 as well as for the commitment to cell death. Taken together, our results demonstrate a molecular sequence of events underlying the mechanism of action of cytotoxicity of LEN or POM in MM cells. Quantitative measurements of Aiolos and Ikaros degradation, and c-Myc and IRF4 downregulation in clinical samples would help validate these findings. Disclosures Bjorklund: Celgene Corp: Employment, Equity Ownership. Havens:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corp: Employment, Equity Ownership. Gandhi:Celgene Corp: Employment, Equity Ownership. Wang:Celgene Corp: Employment, Equity Ownership. Amatangelo:Celgene Corp: Employment, Equity Ownership. Lu:Celgene Corp: Employment. Wang:Celgene Corp: Consultancy. Breider:Celgene Corp: Employment. Ren:Celgene Corp: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Klippel:Celgene Corp: Employment. Chopra:Celgene Corp: Employment, Equity Ownership.

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Effect of the BET Inhibitor, Cpi-0610, Alone and in Combination with Lenalidomide in Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.4255.4255 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4255-4255 ◽

Cited By ~ 7

Author(s):

Ka Tat Siu ◽

Homare Eda ◽

Loredana Santo ◽

Janani Ramachandran ◽

Miroslav Koulnis ◽

...

Keyword(s):

Multiple Myeloma ◽

Protein Expression ◽

Cell Lines ◽

Positive Feedback ◽

Research Funding ◽

Cytotoxic Effect ◽

Feedback Mechanism ◽

Employment Equity ◽

Equity Ownership ◽

Rpmi 8226

Abstract The bromodomain and extraterminal (BET) proteins recognize acetylated lysine residues on histone tails and recruit transcriptional machinery to promote gene expression. The BET proteins are attractive drug targets because they regulate the expression of MYC, BCL2 and NF- κB target genes. We investigated the therapeutic potential of CPI-0610, an inhibitor of BET proteins, currently in Phase I testing in multiple myeloma (MM). Our preliminary data show that human MM cell lines are sensitive to BET inhibition, with IC50 values of 800-1000 nM being observed in MM.1S, MM.1R, RPMI-8226, LR5, H929 and U266 cell lines in 72h culture. We further show that CPI-0610 inhibits MM cell growth in the presence of cytokines and when co-cultured with bone marrow stromal cells. CPI-0610 induces apoptosis and G1 cell cycle arrest associated with MYC downregulation. However, protein levels of BCL2, NF- κ B and MCL1 remain unchanged in MM cells upon BET inhibition. The zinc finger transcriptional factor Ikaros (IKZF1) is highly expressed in MM (GEO dataset GSE36133). It is actively transcribed in the MM.1S cell line with an active transcription start site occupied by BRD4 and MED1 (Loven J et al. Cell 2013). Interestingly, we found that CPI-0610 suppresses Ikaros and IRF4 expression at the levels of both transcription and protein in MM cells. With the use of doxycycline-inducible shRNAs targeting IKZF1, IRF4 and MYC, we identified a positive feedback mechanism that is critical for MM cell survival. Individual knockdown of IRF4, IKZF1 or MYC all lead to induction of apoptosis in MM cells. Suppression of IRF4 decreases both Ikaros and MYC protein expression, suggesting that IRF4 is upstream of both Ikaros and MYC. Downregulation of MYC protein expression is observed following IKZF1 knockdown, suggesting that MYC is downstream of Ikaros. Finally, we observed a decrease in IRF4 protein level upon MYC downregulation, implicating a feedback mechanism from MYC to IRF4. Together, these data illustrate a molecular sequence of events going from IRF4 to IKZF1 to MYC and then back to IRF4, forming a positive feedback loop in MM cells. Based on the observation that shRNA-mediated knockdown of MYC and IKZF1 are toxic to MM, we combined CPI-0610 with lenalidomide, an immunomodulatory drug which stabilizes cereblon and facilitates Ikaros degradation in MM cells (Kronke J et al., and Lu G et al., Science 2014). We observed a synergistic cytotoxic effect in the cell lines tested (MM.1S and RPMI-8226). The enhanced cytotoxic effect of the combined treatment in MM cell lines is due in part to suppression of MYC, IKZF1 and IRF4. Ongoing studies will focus on understanding the molecular mechanism underlying this synergistic combination and validating its efficacy in vivo in order to provide a rationale for clinical protocols of BET inhibitors in MM. Disclosures Mertz: Constellation Pharmaceuticals: Employment, Equity Ownership. Sims:Constellation Pharmaceuticals: Employment, Equity Ownership. Cooper:Constellation Pharmaceuticals: Employment, Equity Ownership. Raje:Celgene Corporation: Consultancy; Eli Lilly: Research Funding; Takeda: Consultancy; Amgen: Consultancy; Onyx: Consultancy; AstraZeneca: Research Funding; Novartis: Consultancy; BMS: Consultancy; Acetylon: Research Funding; Millenium: Consultancy.

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CDK9 Inhibition Reverses Resistance to ABT-199 (GDC-0199) By Down-Regulating MCL-1

Blood ◽

10.1182/blood.v124.21.2161.2161 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2161-2161 ◽

Cited By ~ 1

Author(s):

Jun Chen ◽

Sha Jin ◽

Paul Tapang ◽

Stephen K Tahir ◽

Morey Smith ◽

...

Keyword(s):

Clin Oncol ◽

Cell Lines ◽

Small Molecule ◽

Hodgkin’S Lymphoma ◽

Acquired Resistance ◽

Hematologic Malignancies ◽

Hodgkin's Lymphoma ◽

Employment Equity ◽

Non Hodgkin's Lymphoma ◽

Equity Ownership

Abstract All authors are employees of AbbVie and participated in the design, conduct, and interpretation of these studies. AbbVie and Genentech provided financial support for these studies and participated in the review and approval of this publication. The BCL-2-selective inhibitor ABT-199 has demonstrated efficacy in numerous preclinical models of hematologic malignancies without causing thrombocytopenia, a dose-limiting toxicity associated with the BCL-2/BCL-XL inhibitor navitoclax (Souers et al. 2013. Nat. Med. 19, 202-208). ABT-199 has also demonstrated clinical activity in chronic lymphocytic leukemia (CLL) and non-Hodgkin’s lymphoma (NHL) (Seymour et al. 2014. J. Clin. Oncol. 32, 448s; Davids et al. 2014. J. Clin. Oncol. 32, 544s). Despite these encouraging early clinical data, some subjects do not respond to ABT-199 or progress while on treatment. Pre-clinical models indicate that both intrinsic and acquired resistance may be a consequence of MCL-1 expression. Consequently, we have explored potent and selective small molecule inhibitors of CDK9, a kinase known to maintain the expression of MCL-1 through its role in p-TEFb-mediated transcription. Inhibition of CDK9 resulted in the rapid loss in RNA polymerase II phosphorylation (Serine 5) and MCL-1 expression that was closely followed by the induction of apoptosis in MCL-1-dependent cell lines, a cellular response that could be rescued by overexpression of BCL-2. Substantial synergy was observed between CDK9 inhibitors and ABT-199 in a number of hematologic cell lines with intrinsic or acquired resistance to ABT-199. Direct inhibition of MCL-1 with the small molecule BH3 mimetic A-1210477 was also highly synergistic with ABT-199, further validating the utility of co-inhibiting MCL-1 and BCL-2 function simultaneously in ABT-199 resistant tumors. Importantly, the CDK9 inhibitor-ABT-199 combination was well tolerated in vivo and demonstrated efficacy superior to either agent alone in xenograft models of non-Hodgkin’s lymphoma (NHL) and acute myelogenous leukemia (AML). These data indicate that CDK9 inhibitors may be highly efficacious when used in combination with ABT-199 for the treatment of hematologic malignancies. Disclosures Chen: Abbvie: Employment, Equity Ownership. Jin:Abbvie: Employment, Equity Ownership. Tapang:abbvie: Employment, Equity Ownership. Tahir:abbvie: Employment, Equity Ownership. Smith:abbvie: Employment, Equity Ownership. Xue:abbvie: Employment, Equity Ownership. Zhang:abbvie: Employment, Equity Ownership. Gao:abbvie: Employment, Equity Ownership. Tong:abbvie: Employment, Equity Ownership. Clark:abbvie: Employment, Equity Ownership. Ricker:abbvie: Employment, Equity Ownership. Penning:abbvie: Employment, Equity Ownership. Albert:abbvie: Employment, Equity Ownership. Phillips:abbvie: Employment, Equity Ownership. Souers:abbvie: Employment, Equity Ownership. Leverson:abbvie: Employment, Equity Ownership.

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Hematologic Malignancies Exhibit Selective Vulnerability to Inhibition of De Novo Pyrimidine Biosynthesis By AG-636, a Novel Inhibitor of Dihydroorotate Dehydrogenase in Phase 1 Clinical Trials

Blood ◽

10.1182/blood-2019-123471 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1570-1570

Author(s):

Danielle Ulanet ◽

Victor Chubukov ◽

John Coco ◽

Gabrielle McDonald ◽

Mya Steadman ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Solid Tumor ◽

Research Funding ◽

De Novo ◽

Phase 1 ◽

Cancer Cell Lines ◽

Pyrimidine Biosynthesis ◽

Employment Equity ◽

Equity Ownership

Rapidly proliferating cells reprogram metabolism to support increased biosynthetic demands, a feature that can expose targetable vulnerabilities for therapeutic intervention. A chemical biology screen was performed in an effort to identify metabolic vulnerabilities in particular tumor subtypes, and revealed potent and selective activity of a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic origin. In contrast, cancer cell lines of solid tumor origin exhibited comparatively poor sensitivity. Evaluation of a lymphoma cell line panel demonstrated broad responsiveness to DHODH inhibition, independent of clinical subtype (e.g. ABC, GCB, double-hit). The on-target cellular activity of AG-636 was evaluated by examining the metabolic effects of AG-636 on cells and by evaluating the ability of extracellular uridine to rescue the effects of AG-636 on proliferation and viability. The metabolic changes incurred upon treatment of cells with AG-636 were consistent with a mechanism of action driven by inhibition of DHODH and de novo pyrimidine biosynthesis. Supraphysiologic concentrations of extracellular uridine rescued the effects of AG-636 on growth and viability as well as the effects on metabolism, further confirming on-target activity. The mechanistic basis for differential sensitivity to AG-636 was assessed by comparing the activity of the de novo pyrimidine biosynthesis and uridine salvage pathways in cancer cell lines of hematologic or solid tumor origin with similar proliferative rates. Differential response to AG-636 could not be attributed to varying abilities to utilize the de novo pyrimidine biosynthesis pathway or to salvage extracellular uridine. Real-time imaging of cells treated with AG-636, along with monitoring of extracellular uridine concentrations, demonstrated immediate effects on the viability of lymphoma cell lines in the setting of depleted extracellular uridine. In contrast, solid tumor cell lines were able to maintain growth for an additional period of time, suggestive of adaptive mechanisms to supply pyrimidine pools and/or to cope with nucleotide stress. The high in vitro activity of AG-636 in cancer cells of hematologic origin translated to xenograft models, including an aggressive, patient-derived xenograft model of triple-hit lymphoma and an ibrutinib-resistant model of mantle cell lymphoma in which complete tumor regression occurred. These studies support the development of AG-636 for the treatment of hematologic malignancies. A phase 1 study has been initiated in patients with relapsed/refractory lymphoma (NCT03834584). Disclosures Ulanet: Agios: Employment, Equity Ownership. Chubukov:Agios: Employment, Equity Ownership. Coco:Agios: Employment, Equity Ownership. McDonald:Agios: Employment, Equity Ownership. Steadman:Agios: Employment, Equity Ownership. Narayanaswamy:Agios: Employment, Equity Ownership. Ronseaux:Agios: Employment, Equity Ownership. Choe:Agios: Employment, Equity Ownership. Truskowski:Agios: Employment, Equity Ownership. Nellore:Aurigene Discovery Technologies: Employment. Rao:Firmus Laboratories: Employment, Equity Ownership. Lenz:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Research Funding; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Employment, Honoraria, Research Funding, Speakers Bureau. Cooper:Agios: Employment, Equity Ownership. Murtie:Agios: Employment. Marks:Agios: Employment, Equity Ownership.

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ASLAN003, a Novel and Potent Dihydroorotate Dehydrogenase (DHODH) Inhibitor, Induces Differentiation of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114392 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4047-4047 ◽

Cited By ~ 1

Author(s):

Jianbiao Zhou ◽

Jessie Yiying Quah ◽

Jing Yuan Chooi ◽

Sabrina Hui-Min Toh ◽

Yvonne Ng ◽

...

Keyword(s):

Cell Growth ◽

Cell Lines ◽

Research Funding ◽

Tissue Bank ◽

Dihydroorotate Dehydrogenase ◽

Employment Equity ◽

Healthy Control ◽

Equity Ownership ◽

Nbt Reduction

Abstract Background: Differentiation therapies achieve remarkable success in acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML). However, clinical benefits of differentiation therapies are negligible in non-APL AML, which accounts for the majority of AML cases. Dihydroorotate dehydrogenase (DHODH) regulates the fourth step of the de novo pyrimidine synthesis pathway. DHODH is a key therapeutic target for auto-immune diseases and cancer, particularly differentiation of AML. ASLAN003 is a novel, potent small molecule DHODH inhibitor being developed in AML by ASLAN Pharmaceuticals. Methods: We investigated activity of ASLAN003 in AML cell lines and primary bone marrow (BM) cells (NUS Leukemia Tissue Bank) from patients with AML (N = 14) or myelodysplastic syndromes (MDS) (N = 6) and healthy control (N = 1). We performed CTG assay, FACS analysis of cell viability and myeloid markers, wright-giemsa staining, NBT reduction assay, and qRT-PCR analysis of key lineage transcription factors to evaluate the effects of ASLAN003 on cell growth, differentiation, apoptosis, and gene expression changes in vitro. Two AML cell lines and 1 leukemic patient derived xenograft (PDX) line (NUS Leukemia Tissue Bank) were studied in NSG xenograft mice. Mice were administrated with vehicle control or ASLAN003 50 mg/kg by oral gavage once daily. Results: ASLAN003 inhibited leukemic cell growth of THP-1, MOLM-14 and KG-1 with IC50 of 152, 582 and 382 nM, respectively, at 48 h. Treatment of these leukemia cells with ASLAN003 for 96 h consistently resulted in remarkable increase of CD11b (p < 0.001) and displayed morphologic changes of terminal differentiation and positivity for NBT reduction. ASLAN003 was active in differentiation with an EC50 of 28, 85, and 56 nM, in these 3 lines, respectively. ASLAN003 induced approximately 2-fold higher CD11b+ cells than Brequinar (BRQ), another DHODH inhibitor. Addition of uridine rescued differentiation and improved cell viability in ASLAN003 treated-cells, implying on-target specificity of ASLAN003. Mechanistically, ASLAN003 induced differentiation through induction of myeloid lineage transcription factor Runx1, Pu.1, Gif1 and repression of HoxA9, Gata1. The response of primary BM cells to ASLAN003 was classified into 3 categories: sensitive if any of myeloid markers CD11b, CD14, CD13 or CD33 increased ≥ 15%; moderate: ≥ 5%, but < 15%; resistant: < 5%. Among AML samples, we observed 6 (43%) sensitive cases, 6 (43%) moderate cases and 2 (14%) resistant cases. Three (50%) MDS samples displayed sensitive response and 3 cases (50%) showed moderate response. The healthy control sample was resistant to ASLAN003. Importantly, ASLAN003 promoted differentiation and cell death of myeloid cells in one relapsed AML case. Morphologic analysis and NBT assay demonstrated the features of neutrophil differentiation in selected ASLAN003-treated primary AML blasts. For in vivo experiments, significantly prolonged survival was seen in ASLAN003-treated groups when compared to vehicle control group in both MOLM-14 (p = 0.031) and THP-1 (p < 0.001) xenograft models. ASLAN003 substantially reduced disseminated tumors and leukemic infiltration into liver in xenografted mice. The human CD45+ cells were significantly reduced in BM, peripheral blood, spleen and liver, with significantly increased differentiation of AML cells (CD11b and CD14 positive cells) in BM of treated mice in both models (p < 0.01). We also evaluated the therapeutic efficacy of ASLAN003 in one PDX line, AML-14. At the end of experiments (day 77 post treatment), all PDX mice were alive in both control and ASLAN003 group. The leukemic burden was significantly lower in ASLAN003-treated PDXs than in vehicle-treated PDXs (p = 0.04). Overall, these data demonstrate potent in vivo efficacy of ASLAN003 in inducing myeloid differentiation of blast cells and the drug appears highly tolerable even after prolonged administration. Conclusion: ASLAN003 is a novel, highly potent DHODH inhibitor that induces terminal differentiation, inhibits cell growth and promotes cell death of AML blasts, including relapsed AML blasts. ASLAN003 prolongs survival and shows therapeutic effects in mice bearing different AML cell lines and reduces leukemic burden in an AML PDX model. Currently, ASLAN003 efficacy is being evaluated in a Phase IIa clinical trial in patients with AML (NCT03451084; Ting, ASH abstract 2018). Disclosures Seet: ASLAN Pharmaceuticals: Employment, Equity Ownership. Ooi:ASLAN Pharmaceuticals: Employment, Equity Ownership. Lindmark:ASLAN Pharmaceuticals: Employment, Equity Ownership. McHale:ASLAN Pharmaceuticals: Employment, Equity Ownership. Chng:Amgen: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Aslan: Research Funding; Merck: Research Funding; Janssen: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding; Takeda: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses, Research Funding.

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Combination of Selective Inhibitor of Nuclear Export (SINE) Compounds, Selinexor and KPT-8602, with Venetoclax (ABT-199) Displays Enhanced Activity in Leukemia and Large Cell Lymphoma

Blood ◽

10.1182/blood.v128.22.3949.3949 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3949-3949 ◽

Cited By ~ 3

Author(s):

Melissa A. Fischer ◽

Sharon Friedlander ◽

Leah Hogdal ◽

Pia Arrate ◽

Hua Chang ◽

...

Keyword(s):

Board Of Directors ◽

Cell Lines ◽

Combination Treatment ◽

Nuclear Export ◽

Research Funding ◽

Selective Inhibitor ◽

Employment Equity ◽

Advisory Committees ◽

Cytoplasmic Transport ◽

Equity Ownership

Abstract Introduction: Exportin 1 (XPO1) is a well characterized and essential nucleo-cytoplasmic transport protein in the karyopherin family, and is responsible for the nuclear export of over 200 cargo proteins, including the major tumor suppressor proteins (TSPs) p53, p21, FOXO and the translation regulator elF4E. XPO1 is overexpressed in numerous cancer types including solid and hematological malignancies, often correlating with poor prognosis. Recently, a novel class of Selective Inhibitors of Nuclear Export (SINE) compounds, selinexor (KPT-330) and the second generation KPT-8602, have been developed for the treatment of advanced cancers. We have previously shown that selinexor has marked activity in AML and DLBCL pre-clinical models. The BCL-2 family of anti-apoptotic proteins are deregulated and linked to maintenance and survival in AML and DLBCL. For its translation, the mRNA for BCL-2 is transported from the nucleus to the cytoplasm by forming a complex with XPO1 cargo, elF4E. Other important mRNAs exported from the nucleus via this mechanism include BCL6 and MYC. We hypothesize that SINE compounds inhibit XPO1/elF4E-mediated nuclear-cytoplasmic transport by covalently binding to the XPO1 cargo binding site and that in the absence of protein translation, BCL-2, BCL6 and MYC levels rapidly decline. Venetoclax (VEN; ABT-199) is a potent, selective inhibitor of BCL-2. In vitro, AML cells acquire resistance to VEN over time, often due to up-regulation of another BCL-2 family anti-apoptotic protein, MCL-1. MCL-1 is regulated by the anti-apoptotic transcription factor and XPO1 cargo NF-kB. We have previously shown that SINE compounds significantly decreased MCL-1 levels, presumably via inactivation of NF-kB. The goal of this study was to test whether SINE compounds will synergize with VEN via BCL-2 modulation and whether the combination would diminish MCL-1 mediated resistance to BCL-2 inhibition in DLBCL and AML models, respectively. Methods: BH3 profiling was performed in a sample of cell lines using a cytochrome c release assay to identify anti-apoptotic dependencies. The effects of SINE compounds and VEN as single agents or in combination on cell viability were performed in AML (K-562, MOLM-13, MV-4-11, and U-937) and DLBCL cell lines (SU-DHL-6, DoHH-2 and Toledo). Whole cell protein lysates were extracted 24 hours after treatment for immunoblot analysis. The activity of SINE compounds (5 mg/kg) and VEN (25 mg/kg) as single agents, or in combination were measured in AML (MV-4-11) and DLBCL (DoHH-2 and Toledo) xenografts in NSGS and nude mice, respectively. Tumor growth and survival were measured throughout these animal studies. Tumor tissue was collected at the end of treatment for flow cytometric analysis, western blotting and immunohistochemistry (IHC). Results: By employing BH3 profiling, we identified AML cell lines that were dependent (MV-4-11 and MOLM-13) and not dependent (U-937 and K-562) on MCL-1. Dose response analysis demonstrated that each of the AML cell lines was sensitive to the SINE compounds, while VEN only reduced viability in the MV-4-11 and MOLM-13 cells. Additionally, there was enhanced growth inhibition when the SINE compounds were combined with VEN in the MCL-1 dependent cells. SINE compound treatment synergistically decreased c-MYC protein levels in all 4 AML cell lines with the combination treatment (Figure 1), whereas PARP cleavage was only enhanced with the combination in the MV-4-11 and MOLM-13 cells. Likewise, MCL-1 is reduced in the presence of SINE compound or SINE compound-VEN combinations. In DLBCL xenograft studies (DoHH-2 and Toledo), combination of selinexor with VEN was synergistic for tumor reduction and increased animal survival when compared to either single agent alone. By IHC we observed a concomitant reduction in BCL-2 and BCL-6 and an increase in cleaved caspase 3 in DLBCL tumors after combination treatment. Conclusions: SINE compound-VEN combinations show enhanced antitumor effect, with reduction of oncogenic activity. SINE compounds reduce MCL-1 in VEN-resistant cells. As MCL-1 driven anti-apoptotic machinery is responsible for resistance to inhibition of BCL-2 in DLBCL and AML, SINE compound regulation of MCL-1 may lead to rescue of VEN resistance. SINE compounds and VEN are excellent candidate partners for combination therapies in AML and DLBCL. Disclosures Friedlander: Karyopharm Therapeutics: Employment. Chang:Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Argueta:Karyopharm Therapeutics: Employment, Equity Ownership. Klebanov:Karyopharm Therapeutics: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics: Employment, Equity Ownership. Baloglu:Karyopharm Therapeutics: Employment, Equity Ownership. Lee:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Savona:TG Therapeutics: Research Funding; Amgen Inc.: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Sunesis: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

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BH3 Profiling Predicts 5-Azacytidine Response in Malignant Myeloid Cells

Blood ◽

10.1182/blood.v120.21.1432.1432 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1432-1432

Author(s):

James M Bogenberger ◽

William E. Pierceall ◽

Ryan Lena ◽

Reinhold Munker ◽

Kaoru Tohyama ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cell Population ◽

Research Funding ◽

Family Members ◽

Myeloid Cells ◽

Functional Assay ◽

Employment Equity ◽

Mitochondrial Outer Membrane Permeabilization ◽

Equity Ownership

Abstract Abstract 1432 Previously we demonstrated that inhibition of anti-apoptotic BCL-2 family members sensitize leukemic cells to 5-Azacytidine (5-Aza), using siRNA and pharmacological inhibition with the BH3-mimetic ABT-737, both in vitro and ex vivo (Bogenberger, JM., et. al. ASH Annual Abstracts 2011;118:Abstract 3513). Crucially, several anti-apoptotic BCL-2 members (e.g. BCL-2 and BCL-XL) required concurrent inhibition for potent and universal sensitization to 5-Aza. Anti-apoptotic BCL-2 proteins block the execution of programmed cell death (apoptosis) by binding to and counteracting two types of pro-apoptotic BCL-2 family proteins: the “BH3-only” proteins, including both activators (BIM and BID) and sensitizers (e.g. NOXA, PUMA, HRK), and the multi-domain effector proteins (BAX and BAK). Cells dependent on anti-apoptotic BCL-2 family members for survival have been defined as “primed for death” (Certo, M., et. al. Cancer Cell 2006 May;9(5):351-65). Importantly, the priming status reflects a dependence on anti-apoptotic BCL-2 family proteins and can be characterized with the BH3 profiling functional assay. This assay measures induction of mitochondrial outer membrane permeabilization (MOMP) in response to treatment with peptides derived from BH3-only proteins (Ni Chonghaile, T., et. al. Science 2011 Nov 25;334(6059);1129-33). Thus, the unique BH3 profile associated with a specific malignant cell population, is a function of the anti-apoptotic BCL-2 family member/s contextual expression in that cell population. Based on our observations of potent 5-Aza sensitization in combination with anti-apoptotic BCL-2 protein family inhibition, we hypothesized that BH3-profiling would predict response to 5-Aza. To address the potential utility of BH3 profiling in predicting response to 5-Aza in myeloid malignancies, we assayed a broad panel of AML-derived cell lines (N=13) by BH3 profiling and correlated BH3 metrics with 5-Aza response. Identical cell line passages were assayed by BH3 profiling and in 5-Aza drug dose response assays. The cell panel comprised lines derived from AML FAB subtypes M7, M6, M5, M4, M2 and M0, as well as diverse cytogenetics such as t(11;21), t(9;11), t(4;11), t(6;11), del 5q, del 7, and a broad spectrum of mutations such as FLT3, N-RAS, CDKN2A, NPM1 and DNMT3A. The panel included a normal karyotype AML cell line (CG-SH) (Munker, R., et. al. Leuk Res 2009 Oct:33(10):1405-8) and a blastic cell line derived from a patient with MDS (MDS-L) (Tohyama, K., et. al. Br J Haematol 1994 Jun;87(2):235-42). Partition modeling using several BH3 metrics discriminates 5-Aza response with statistical significance (N=13, Mann-Whitney p<0.01) between more sensitive (EC50<2uM) and less sensitive (EC50>2uM) AML lines. For instance, the Puma BH3 peptide alone distinguishes more sensitive from less sensitive cell lines (N=13, Mann-Whitney p=0.0046). Several more complex parameters, such as “Puma+Hrk”, “Puma+Noxa,” and “Bim+Puma+Noxa+Hrk,” also significantly discriminate 5-Aza response (Mann-Whitney p=0.0011). Using continuous variable analysis, a Puma+Hrk metric exhibited the strongest correlation with 5-Aza response (R2=0.85, p<0.0001), while an individual Puma metric yielded the second strongest correlation (R2=0.70, p=0.0004). In conclusion, BH3 profiling discriminates 5-Aza response and confirms a central role for anti-apoptotic BCL-2 members in 5-Aza response. Furthermore, HRK priming indicates that BCL-XL is an important, although not the sole, anti-apoptotic component determining 5-Aza response in myeloid cells. These BH3 profiling results are in agreement with our functional 5-Aza RNAi and BCL-XL/BCL-2 inhibitor data in combination with 5-Aza. Based on these results, we are currently evaluating BH3 profiles from 5-Aza responding and non-responding AML and MDS patients to validate BH3 profiling-derived metrics in predicting clinical outcomes to 5-Aza. Disclosures: Pierceall: Eutropics Pharmaceuticals: Employment, Equity Ownership. Lena:Eutropics Pharmaceuticals: Employment. Mesa:Sanofi: Research Funding; Incyte: Research Funding; Lilly: Research Funding; NS Pharma: Research Funding; YM Bioscience: Research Funding. Cardone:Eutropics Pharmaceuticals: Employment, Equity Ownership.

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