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

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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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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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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Eltrombopag Decreases Proliferation of Ovarian, Lung and Breast Tumor Cell Lines.

Blood ◽

10.1182/blood.v114.22.2409.2409 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2409-2409

Author(s):

Connie L. Erickson-Miller ◽

Jennifer Kirchner ◽

Kodandaram Pillarisetti ◽

Lone Ottesen ◽

Yasser Mostafa Kamel ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Cell ◽

Cell Lines ◽

Mrna Levels ◽

Tumor Cell Lines ◽

Employment Equity ◽

Qrt Pcr ◽

Thrombopoietin Receptor ◽

Equity Ownership ◽

Low Levels

Abstract Abstract 2409 Poster Board II-386 Background: Eltrombopag (Promacta®) is a novel, oral thrombopoietin receptor (TpoR) agonist that interacts with the TpoR on bone marrow progenitors to stimulate megakaryocyte production, thus increasing platelet counts in thrombocytopenic patients. The effects of eltrombopag on the proliferation of solid tumor cell lines and the expression of thrombopoietin receptor (MPL, TpoR) on patient tumors is of interest given that chemotherapy can cause thrombocytopenia. Materials and methods: Proliferation was measured by Cell Titer Glo assay on 3 ovarian (OVCAR3, OVCAR4, SKOV3), 4 lung (A549, NCI-H226, NCI-H510, NCI-H460) and 3 breast (BT-474, MCF7, HCC1937) cancer cell lines from the ATCC treated with 0.01 – 100 ug/mL eltrombopag. Quantitative RT-PCR (qRT-PCR) for MPL expression was performed on the tumor cell lines and on 40 tumor samples, each from subjects with ovarian, lung or breast cancer. Microarray analysis for MPL mRNA expression was examined from 118 subjects with breast cancer and 29 with non-small cell lung cancer (NSCLC). Microarray data was normalized using robust multiarray average (RMA) and relative mRNA expression was determined. To determine expression of TpoR protein, western blot analyses was performed on some of the tumor cell lines. Results: Eltrombopag induced an inhibition of proliferation on all of the ovarian, lung and breast solid tumor cell lines tested. The IC50 ranged from 3.7 to 49.7 ug/mL (see table below). The Cmax of ITP patients treated with 75 mg eltrombopag is 11.4 ug/mL, demonstrating that these concentrations are clinically achievable. There was no enhancement of proliferation at any concentration of eltrombopag, consistent with the very low or undetectable level of MPL expression on samples of tumors from patients with these diseases. MPL was expressed at very low or undetectable levels in these tumor cell lines with the exception of the lung cancer line, NCI-H510. However, western blot analyses showed no detectable TpoR protein expression regardless of the higher levels of MPL mRNA in NCI-H510 cells. Erythropoietin receptor (EPOR) mRNA was expressed at low-to-moderate levels, while ERBB2 and IGF1R were expressed at higher levels in these cell lines. Microarray analysis showed undetectable MPL mRNA levels in all 118 samples from patients with breast cancer and 52% of the NSCLC samples, the remaining NSCLC samples expressed low levels of MPL. In contrast, EPOR was expressed in 75–100% of the breast cancer, and NSCLC samples. ERBB2 was expressed in 97–100% of the samples and IGF1R was expressed in 54–100% of the samples. When 40 other tumor samples each from subjects with ovarian, lung and breast cancer were examined by qRT-PCR, MPL mRNA levels were also very low or undetectable. EPOR, ERBB2, and IGF1R expression levels varied according to tumor type, but were greater than MPL levels. Conclusions: In summary, similar to its effects on leukemia and lymphoma cell lines, all of the nine lung, ovarian, breast or prostate tumor cell lines demonstrated decreased proliferation in response to eltrombopag. The undetectable or very low levels of expression of MPL mRNA in tumors of patients with lung, ovarian, breast or prostate cancer supports the proliferation results. Disclosures: Erickson-Miller: GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties, Research Funding. Kirchner:GlaxoSmithKline: Employment. Pillarisetti:GSK: Employment, Equity Ownership, Patents & Royalties. Ottesen:GSK: Employment, Equity Ownership. Mostafa Kamel:GSK: Employment, Equity Ownership. Liu:GSK: Employment, Equity Ownership. Martin:GSK: Employment, Equity Ownership. Messam:GSK: Employment, Equity Ownership.

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Inhibition Of Nuclear Transport Modulator Xpo1/CRM1 For The Treatment Of Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v122.21.237.237 ◽

2013 ◽

Vol 122 (21) ◽

pp. 237-237 ◽

Cited By ~ 2

Author(s):

Michael P. Rettig ◽

Matthew Holt ◽

Julie Prior ◽

Sharon Shacham ◽

Michael Kauffman ◽

...

Keyword(s):

Flow Cytometry ◽

Board Of Directors ◽

Cell Lines ◽

Nuclear Export ◽

Employment Equity ◽

Advisory Committees ◽

Ic50 Values ◽

Equity Ownership

Abstract Background Exportin 1 (XPO1) also called CRM1, is a widely expressed nuclear export protein, transporting a variety of molecules including tumor suppressor proteins and cell cycle regulators. Targeted inhibition of XPO1 is a new strategy to restore multiple cell death pathways in various malignant diseases. SINEs are novel, orally available, small molecule Selective Inhibitors of Nuclear Export (SINE) that specifically bind to XPO1 and inhibit its function. Methods We used WST-1 cell proliferation assays, flow cytometry, and bioluminescence imaging to evaluate the efficacy of multiple SINEs to induce apoptosis alone and in combination with cytarabine (AraC) or doxorubicin in vitro in chemotherapy sensitive and resistant murine acute promyelocytic leukemia (APL) cells. This murine model of APL was previously generated by knocking in the human PML-RARa cDNA into the 5’ regulatory sequence of the cathepsin G locus (Westervelt et al. Blood, 2003). The abnormal co-expression of the myeloid surface antigen Gr1 and the early hematopoietic markers CD34 and CD117 identify leukemic blasts. These Gr1+CD34+CD117+ APL cells partially retain the ability to terminally differentiate toward mature granulocytes (mimicking more traditional AML models) and can be adoptively transferred to secondary recipients, which develop a rapidly fatal leukemia within 3 weeks after tumor inoculation. To assess the safety and efficacy of SINEs in vivo, we injected cryopreserved APL cells intravenously via the tail vein into unconditioned genetically compatible C57BL/6 recipients and treated leukemic and non-leukemic mice (n=15/cohort) with 15 mg/kg of the oral clinical staged SINE KPT-330 (currently in Phase 1 studies in patients with solid tumors and hematological malignancies) alone or in combination with 200 mg/kg cytarabine every other day for a total of 2 weeks. Peripheral blood was obtained weekly from mice for complete blood counts and flow cytometry to screen for development of APL. Results The first generation SINE, KPT214, inhibited the proliferation of murine APL cell lines in a dose and time dependent manner with IC50 values ranging from of 95 nM to 750 nM. IC50 values decreased 2.4-fold (KPT-185) and 3.5-fold (KPT-249) with subsequent generations of the SINEs. Consistent with the WST-1 results, Annexin V/7-aminoactinomycin D flow cytometry showed a significant increase of APL apoptosis within 6 hours of KPT-249 application. Minimal toxicity against normal murine lymphocytes was observed with SINEs even up to doses of 500 nM. Additional WST-1 assays using AraC-resistant and doxorubicin-resistant APL cell lines demonstrated cell death of both chemotherapy-resistant cell lines at levels comparable to the parental chemosensitive APL cell lines. Combination therapy with low dose KPT-330 and AraC showed additive effects on inhibition of cell proliferation in vitro. This additive effect of KPT-330 and chemotherapy on APL killing was maintained in vivo. As shown in Figure 1, treatment with AraC or KPT-330 alone significantly prolonged the survival of leukemic mice from a median survival of 24 days (APL + vehicle) to 33 days or 39 days, respectively (P < 0.0001). Encouragingly, combination therapy with AraC + KPT-330 further prolonged survival compared to monotherapy (P < 0.0001), with some mice being cured of the disease. Similar in vivo studies with the AraC-resistant and doxorubicin-resistant APL cells are just being initiated. Conclusions Our data suggests that the addition of a CRM1 inhibitor to a chemotherapy regimen offers a promising avenue for treatment of AML. Disclosures: Shacham: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. McCauley:Karyopharm Therapeutics, Inc: Employment, Equity Ownership.

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Iberdomide (CC-220) Has Synergistic Anti-Tumor and Immunostimulatory Activity Against Multiple Myeloma in Combination with Both Bortezomib and Dexamethasone, or in Combination with Daratumumab in Vitro

Blood ◽

10.1182/blood-2018-99-113383 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1935-1935 ◽

Cited By ~ 7

Author(s):

Michael Amatangelo ◽

Chad C. Bjorklund ◽

Jian Kang ◽

Ann Polonskaia ◽

Sridevi Viswanatha ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Cell Lines ◽

Immune Cells ◽

Antiproliferative Activity ◽

Mononuclear Cells ◽

Interleukin 2 ◽

Immunomodulatory Activity ◽

Employment Equity ◽

Equity Ownership

Abstract Despite significant progress in the treatment of multiple myeloma (MM), the disease remains incurable. Multiple targeted and biologics-based therapies, including immunomodulatory agent IMiD® compounds (lenalidomide and pomalidomide), proteasome inhibitors (bortezomib and carfilzomib), and monoclonal antibodies (daratumumab and elotuzumab) have shown impressive activity in treating advanced MM. Moreover, triplet regimens combining these agents have consistently proven to be more efficacious than doublets in heavily pretreated patients with limited additional toxic effects. Iberdomide (CC-220) is a novel compound being investigated in a phase I/II study (clinicaltrials.gov NCT02773030) for treatment of lenalidomide- and pomalidomide-relapsed/refractory MM (RRMM) in combination with dexamethasone. Preclinical studies of iberdomide have shown that it more potently binds to cereblon than other cereblon-binding compounds, is more efficient at degrading Aiolos and Ikaros, and has enhanced immunomodulatory activity, inducing greater interleukin-2 secretion and granzyme-b degranulation in immune cells (Matyskiela et al and Bjorklund et al, submitted abstract). Clinical studies of bortezomib and daratumumab in combination with other cereblon-binding agents have demonstrated high tolerability with notable efficacy in the RRMM setting; however, these combinations with iberdomide have not been investigated. Here we show in MM cell lines that iberdomide induces deep Aiolos and Ikaros degradation in the presence of bortezomib at clinically relevant concentrations as determined in healthy volunteers. Furthermore, iberdomide treatment in combination with bortezomib produced synergistic antiproliferative activity and deeper induction of apoptosis than combinations of other clinically approved cereblon-binding compounds with bortezomib across multiple cell lines. In addition, adding dexamethasone resulted in further synergistic antiproliferative activity. In combination with daratumumab, iberdomide also had synergistic anti-MM activity in Complement-Dependent Cytotoxicity (CDC) assays. In co-culture systems using myeloma and immune cells, iberdomide significantly increased the antibody-dependent cellular cytotoxic activity of daratumumab. While iberdomide treatment of MM cell lines resulted in increased CD38 surface expression, combinations were more effective when peripheral blood mononuclear cells (PBMCs) were pretreated, suggesting that iberdomide immunomodulatory activity is a significant contributor to the synergy observed. Interestingly, pretreatment of PBMCs with daratumumab resulted in reduced efficacy of the combination. We observed that the treatment of PBMCs with daratumumab resulted in killing of natural killer (NK) cells in the PBMC culture. In contrast, treatment of PBMCs with iberdomide resulted in proliferation of NK cells, possibly helping to rescue the antagonistic effect of daratumumab on NK cell-mediated antibody‐dependent cellular cytotoxicity. Taken together, these preclinical data support further investigation of iberdomide in combination with both bortezomib/dexamethasone and daratumumab in the clinic. Disclosures Amatangelo: Celgene Corporation: Employment, Equity Ownership. Bjorklund:Celgene Corporation: Employment, Equity Ownership. Kang:Celgene Corporation: Employment, Equity Ownership. Polonskaia:Celgene Corporation: Employment, Equity Ownership. Viswanatha:Celgene Corporation: Employment, Equity Ownership. Thakurta:Celgene Corporation: Employment, Equity Ownership.

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CC-92480, a Novel Cereblon E3 Ligase Modulator, Is Synergistic with Dexamethasone, Bortezomib, and Daratumumab in Multiple Myeloma

Blood ◽

10.1182/blood-2019-124345 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1815-1815

Author(s):

Lilly Wong ◽

Rama Krishna Narla ◽

Jim Leisten ◽

Daniel Bauer ◽

Matthew Groza ◽

...

Keyword(s):

Multiple Myeloma ◽

Synergistic Effect ◽

Cell Viability ◽

Cell Lines ◽

E3 Ligase ◽

Proteasome Activity ◽

Employment Equity ◽

Equity Ownership

Introduction: CC-92480 is a novel cereblon E3 ligase modulator (CELMoD) with enhanced autonomous cell-killing and immunomodulatory activity against multiple myeloma (MM) cells. CC-92480 is currently in phase 1 development in a late-line myeloma patient population (NCT03374085). Here, we sought to characterize the antitumor activity of CC-92480 in combination with dexamethasone (DEX), bortezomib (BORT), or daratumumab (DARA) in MM cell lines in vitro and xenograft mouse models in vivo. Methods: CC-92480 activity in combination with DEX was evaluated in MM cell lines. Apoptosis was measured by quantification of caspase-3 activation. The effect of BORT on CC-92480-induced Ikaros and Aiolos degradation was determined by concurrent treatment of MM cells with BORT and CC-92480. β5-site proteasome activity was also determined in the same experiment. The in vitro activity of CC-92480 in combination with BORT was characterized using washout experiments to more faithfully model the short in vivo exposure but more prolonged, gradually diminishing proteasome inhibitory activity of BORT. Apoptosis and cell viability of CC-92480 with BORT were analyzed by flow cytometry. The effect of CC-92480 on CD38 expression was also evaluated across a panel of MM cell lines. The effect of CC-92480 in combination with DARA was characterized with antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) assays. CC-92480 in combination with DEX or BORT was tested in a lenalidomide-resistant (H929-1051) xenograft mouse model. Female SCID mice were inoculated with H929-1051 cells in the right hind leg. For the DEX combination, groups of tumor-bearing mice (n = 9-10) were dosed with vehicle, DEX, or CC-92480 once daily (QD), or CC-92480 in combination with DEX throughout the study, starting when the tumor volumes reached approximately 115 mm3. For combination with BORT, mice (n = 9-10/group) were dosed with vehicle, CC-92480, or BORT, or the CC-92480 and BORT combination starting when the tumor volumes reached approximately 500 mm3. CC-92480 was administered orally QD for 3 days and BORT as a single intravenous dose. Tumor volumes were measured twice a week for the duration of the studies. Results: CC-92480 synergized with DEX in reducing cell viability and potentiated DEX-induced apoptosis in a concentration-dependent manner in MM cell lines. Of note, the combination showed activity at concentrations of both DEX and CC-92480 that had minimal activity as single agents. In the xenograft model with H929-1051 cells, the combination of CC-92480 and DEX significantly inhibited tumor growth (−84%) when compared with either agent alone (−34% and −20% for CC-92480 and DEX, respectively) and was classified as a synergistic effect using the fractional product method. Although proteasome activity is required for CC-92480-induced degradation of Ikaros and Aiolos, CC-92480 nevertheless maintained its ability to efficiently degrade Ikaros and Aiolos in the presence of doses of BORT that cause clinically relevant levels of proteasome inhibition. The in vitro combination of CC-92480 with BORT resulted in greater cytotoxic activity on MM cells than either single agent alone. The in vivo efficacy of CC-92480 and BORT, administered concurrently, showed a strongly synergistic effect with a near complete or complete tumor regression in every animal, and 6 of 9 animals remained tumor-free through an observation period extending 157 days after the control group was terminated. Anti-CD38 therapies, including DARA and isatuxumab, target CD38-expressing MM cells for killing by immune cells through cytotoxic and phagocytic mechanisms. In a panel of MM cell lines, CC-92480 treatment caused increased cell surface expression of CD38 (2-3 times that of control). Pretreatment of MM cells with CC-92480 resulted in increased DARA-mediated ADCC and ADCP compared with DMSO-treated controls. Conclusions: The strong preclinical synergy in MM cell killing exhibited by CC-92480 in combination with DEX, BORT, and with an anti-CD38 antibody (DARA), highlights its potential to bring clinical benefit to patients with MM in combination with these agents and supports the rationale for testing these combinations in clinical studies. Disclosures Wong: Celgene Corporation: Employment, Equity Ownership. Narla:Celgene Corporation: Employment, Equity Ownership. Leisten:Celgene Corporation: Employment. Bauer:Celgene Corporation: Employment, Equity Ownership. Groza:Celgene Corporation: Employment, Equity Ownership. Gaffney:Celgene: Employment. Havens:Celgene: Equity Ownership; Pfizer: Employment, Equity Ownership. Choi:AnaptysBio Inc: Employment, Equity Ownership; Celgene Corporation: Equity Ownership, Other: Formerly Employed. Lopez-Girona:Celgene Corporation: Employment. Hansen:Celgene Corporation: Employment. Cathers:Celgene Corporation: Equity Ownership; Global Blood Therapeutics (GBT): Employment. Carmichael:Celgene plc: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership.

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Protides of 5-Fluorotroxacitabine Display Potent Anti-Proliferative Properties and Circumvent Deoxycytidine Kinase-Mediated Resistance Associated with Cytotoxic Cytidine Analogues; A Novel Approach for Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-114440 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3497-3497

Author(s):

Pedro Pinho ◽

Helen Kylefjord ◽

Vilma Rraklli ◽

Christina Rydergård ◽

Biljana Rizoska ◽

...

Keyword(s):

Cell Lines ◽

Cytidine Deaminase ◽

B Cell Lymphoma ◽

Similar Efficacy ◽

Deoxycytidine Kinase ◽

Employment Equity ◽

Treatment Regimens ◽

Ic50 Values ◽

Equity Ownership

Abstract The cytotoxic nucleoside cytarabine forms the backbone of AML induction and consolidation therapies, but is associated with severe toxicities that preclude its use in patients unable to tolerate aggressive chemotherapy. Options for patients that do not respond to cytarabine, or relapse post-treatment, are limited. Elderly patients and those with relapsed/refractory AML would particularly benefit from the availability of new agents to develop treatment regimens that provide increased efficacy and tolerability compared to cytarabine, and that have a decreased susceptibility to mechanisms of cytarabine resistance, such as decreased deoxycytidine kinase (dCK) and/or upregulation of cytidine deaminase (CDA). Our preclinical evaluation of potential new anti-proliferative chemotherapeutics identified 5-fluorotroxacitabine (5FTRX), a chain-terminating cytidine-based L-nucleoside, as having promising anti-proliferative activity against AML cell lines, and resistance to degradation by CDA. To understand potential mechanisms of resistance to 5FTRX, we selected a population of THP1 (THP1-R) cells resistant to 5FTRX. THP1-R cells were 66-fold resistant to 5FTRX and cross resistant to cytarabine (35-fold) with CC50 values for both nucleosides >50 μM. We discovered that THP1-R cells had decreased levels of dCK (>95% by Western blot), the kinase responsible for the phosphorylation of cytidine and cytidine analogues such as troxacitabine and cytarabine to their corresponding monophosphates. Confirming the importance of dCK in the activation of 5FTRX and cytarabine, chemical inhibition of dCK also rendered THP1 cells >90-fold resistant to 5FTRX and cytarabine. To develop molecules that overcome resistance to both high CDA and low dCK, we used protide technology to construct nucleotide monophosphate prodrugs of 5FTRX, including one potent example, MV806. MV806 was not dependent upon dCK as it maintained similar efficacy in THP1-R cells with low dCK and against THP1 cells treated with the selective dCK inhibitor. We tested MV806 and 5FTRX in a panel of AML cell lines (n=7). MV806 was more potent than 5FTRX with CC50 values ranging from 0.0020-0.19 μM, compared to 0.057-1.2 μM for 5FTRX. MV806 also demonstrated CC50s <0.1 μM against selected T- and B-cell lymphoma cell lines (e.g. MOLT4 and RAJI). Increased in vitro potency of this prodrug compared to 5FTRX correlated with elevated intracellular triphosphate levels in AML cells; MV806 generated 5-fold more triphosphate than 5FTRX in MV4-11 cells. We also tested MV806 in combination with doxorubicin or azacytidine in two AML cell lines (MV4-11 and THP-1). In both tested cell lines, strong synergy was observed (Bliss independence analysis synergy volumes >100), demonstrating future opportunities for clinical combinations. Finally, we showed that MV806 had DMPK profiles suitable for preclinical and clinical development. Leading protides were highly soluble, had a predicted half-life of >6h in human blood and demonstrated IC50 values >1 μM against major CYP isoforms (2A6, 2C9, 2D6, 3A4) with no evidence of time-dependent inhibition at 1 μM. To conclude, we used protide technology to directly deliver the active monophosphate species of 5FTRX intracellularly and thereby overcome resistance to cytarabine due to down-regulation of dCK and increased CDA expression. Taken together, our findings support the further development of protides of 5FTRX for the treatment of AML, including AML patients with reduced sensitivity to cytarabine through high CDA expression and/or low dCK expression. Disclosures Pinho: Medivir AB: Employment, Equity Ownership. Kylefjord:Medivir AB: Employment, Equity Ownership. Rraklli:Medivir AB: Employment. Rydergård:Medivir AB: Employment, Equity Ownership. Rizoska:Medivir AB: Employment, Equity Ownership. Eneroth:Medivir AB: Employment, Equity Ownership. Bylund:Medivir AB: Employment, Equity Ownership. Moses:Medivir AB: Employment, Equity Ownership. Norin:Medivir AB: Employment, Equity Ownership. Bethell:Medivir AB: Employment, Equity Ownership. Schimmer:Otsuka Pharmaceuticals: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Consultancy; Medivir AB: Research Funding. Albertella:Medivir AB: Employment, Equity Ownership. Targett-Adams:Medivir AB: Employment, Equity Ownership.

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Elucidating the Mechanism of Action of CC-90009, a Novel Cereblon E3 Ligase Modulator, in AML Via Genome-Wide CRISPR Screen

Blood ◽

10.1182/blood-2019-125492 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 405-405 ◽

Cited By ~ 2

Author(s):

Gang Lu ◽

Christine Surka ◽

Chin-Chun Lu ◽

In Sock Jang ◽

Kai Wang ◽

...

Keyword(s):

Cell Lines ◽

Mechanism Of Action ◽

E3 Ligase ◽

Inhibitory Effect ◽

Growth Inhibitory Effect ◽

Employment Equity ◽

Growth Inhibitory ◽

Genome Wide ◽

A Genome ◽

Equity Ownership

CC-90009 is a novel cereblon E3 ligase modulator (CELMoD) currently under investigation in a phase I clinical study in relapsed or refractory acute myeloid leukemia (R/R AML) (CC-90009-AML-001; NCT02848001). CC-90009 coopts the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex to target the translation termination factor G1 to S phase transition 1 (GSPT1) for ubiquitination and proteasomal degradation, resulting in rapid induction of apoptosis and growth inhibition in AML cell lines and primary patient blasts. To further elucidate the mechanism of action of CC-90009 in AML, we performed a genome-wide CRISPR/Cas9 screen to identify gene(s) whose knockout abrogate(s) the response to CC-90009 in a sensitive AML cell line. In addition to well-established key regulatory proteins required for the activity of all known cereblon modulators, which include components of the CRL4CRBN complex, E2 ubiquitin conjugating enzymes UBE2G1 and UBE2D3, and members of the neddylation and deneddylation machinery, interestingly, the screen identified the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreased the production of full-length CRBN transcript via modulating alternative splicing of CRBN mRNA, leading to significant downregulation of cereblon expression and hence diminished response to CC-90009. The screen also revealed that mTOR signaling and the integrated stress response (ISR) specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Since CC-90009 inhibits protein translation, it is reasonable to expect interactions with regulators of this pathway. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 , at least in part by reducing CC-90009 induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Loss of GCN2 significantly attenuated the growth inhibitory effect of CC-90009, and this effect can be rescued with GCN2 wild-type but not enzymatically-dead mutants. Collectively, the antitumor activity of CC-90009, a first-in-class GSPT1 degrader, in AML cell lines is mediated by multiple layers of signaling networks and machinery, the elucidation of which reveals the underlying mechanism by which CC-90009 exerts its anti-AML activity and informs on the pathways for further study of CC-90009's clinical utility. Disclosures Lu: Celgene Corporation: Employment, Equity Ownership. Surka:Celgene: Employment, Equity Ownership. Lu:Celgene Corporation: Employment, Equity Ownership. Jang:Celgene: Employment, Equity Ownership. Wang:Celgene: Employment, Equity Ownership. Rolfe:Celgene: Employment, Equity Ownership.

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A Novel Full Length Anti-FLT3 CD3 Bispecific Antibody for the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2018-99-119915 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1445-1445 ◽

Cited By ~ 1

Author(s):

Ivana Djuretic ◽

Veena Krishnamoorthy ◽

Cesar Sommer ◽

Danielle E. Dettling ◽

Kris Poulsen ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

Dendritic Cells ◽

Cell Lines ◽

Bispecific Antibody ◽

Employment Equity ◽

Equity Ownership ◽

Low Levels ◽

Anti Tumor Activity

Abstract FLT3 is a receptor tyrosine kinase expressed on the surface of acute myeloid leukemia (AML) patient blasts. FLT3 is the most frequently mutated gene in AML patients, and these mutations are associated with poor prognosis. Despite the development of small molecule inhibitors of FLT3 function and neutralizing FLT3 antibodies, there remains a need for antibodies that target the broad AML patient population with improved efficacy and safety. We chose to use Pfizer's proprietary full length humanized CD3 bispecific IgG molecule platform. The first step in the development was finding a suitable targeting epitope on FLT3 as not all epitopes result in optimal T cell activation in the context of CD3 binding. Through a combination of in vitro and in vivo studies, FLT3 antibodies targeting extracellular domain 4 of FLT3 were found to be more effective at AML cell depletion than other domains in the full-length bispecific IgG format, significantly outperforming the antibodies targeting the most membrane proximal region of domain 5. The final candidate antibody was engineered to have picomolar affinity for recombinant human FLT3 (<50 pM) to ensure that low levels of FLT3 present on AML (<5,000 per cell) could efficiently trigger T cell cytotoxicity. Anti-tumor activity of the final molecule was further characterized in vitro and in vivo. Three AML cell lines, Eol-1, Molm-13 and MV-411 express high, medium and low levels of FLT3, respectively, and were targeted by activated healthy donor T cells in the presence of the FLT3 bispecific with an efficiency that correlated with FLT3 surface density (EC50 range was 0.5 pM to 40 pM at E:T ratios of 1:1). Ability to target primary AML cells was confirmed in assays with primary AML blasts and autologous patient T cells (EC50s 7 - 15 nM, for E:T ratios of up to 1:50). In orthotopic xenograft models with the same set of cell lines, all three cell lines were efficiently eliminated by activated T cells in the presence of a single dose of FLT3 bispecific (dose range 10 to 100 μg/kg), confirming the anti-tumor activity of the bispecific antibody in vivo. The safety of Pfizer's FLT3 bispecific antibody was also studied in preclinical studies. We first analyzed gene expression of FLT3 and three other commonly considered AML targets (CD33, CD123, and CLL-1) in publicly available gene expression databases. FLT3 had the highest differential expression between AML and healthy tissues with blood and brain showing expression of low levels of FLT3 RNA. In tissue cross-reactivity studies, we detected little to no binding of the candidate FLT3 antibody to human brain tissue sections. In blood, the highest expression of FLT3 was in hematopoietic stem cells and progenitors, dendritic cells and monocytes consistent with previous publications and known roles of FLT3 in hematopoiesis and dendritic cell homeostasis. Of note, expression of FLT3 in monocytes and whole blood was the lowest compared to three other AML targets. To address any potential toxicities of FLT3 bispecific, we performed exploratory studies in cynomolgus monkeys. Affinity of FLT3 bispecific for cynomolgus FLT3 was ~20x lower than for human FLT3 (~1 nM), as determined in binding assays with cells expressing human and cynomolgus FLT3. No major clinical signs or toxicological findings were observed up to the highest dose level tested (3 mg/kg) following two administrations at weekly intervals. On target activity was demonstrated by nearly complete elimination of FLT3+ dendritic cells in the blood of treated monkeys two days after administration. In addition, we detected elimination of FLT3+ CD34+ stem cells in the bone marrow of treated monkeys two days after the second dose. Remarkably, both dendritic cells and FLT3+ CD34+ stem cells rebounded to baseline levels observed in control animals when analyzed at study termination, two weeks following the second dose. Consistent with minimal expression of FLT3 on healthy tissues and the presence of FLT3+ on rare blood subsets, cytokine increases typically associated with CD3 bispecific administration were minimal (only 6 to 17 and 46 to 89 fold over baseline for IFN-γ and IL-6, respectively, at the highest dose tested). In conclusion, the robust anti-tumor activity of Pfizer's FLT3 bispecific antibody combined with good tolerability in cynomolgus monkeys, reversible hematological toxicity and absence of non-hematological toxicity support its further clinical development in AML. Disclosures Djuretic: Pfizer Inc.: Employment. Krishnamoorthy:Pfizer: Employment. Sommer:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Dettling:Maverick Therapeutics: Employment. Poulsen:Allogene Therapeutics: Employment, Equity Ownership. Chen:Pfizer: Employment. Hu:Pfizer: Employment. Given Chunyk:Pfizer: Employment. Lindquist:Pfizer: Employment. Potluri:Pfizer: Employment. Rickert:Pfizer: Employment. Sasu:Allogene Therapeutics: Employment, Equity Ownership, Patents & Royalties. Chaparro-Riggers:Pfizer Inc.: Employment, Patents & Royalties. Yeung:Pfizer: Employment, Patents & Royalties.

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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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