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Baseline (screening) vs treatment cycle 2 (P=.06; paired t test). For patient 5, baseline data were not available.

Purpose:Assess safety and efficacy of nivolumab plus nab-paclitaxel and gemcitabine in patients with locally advanced/metastatic pancreatic cancer in a two-part, open-label, phase I trial.Patients and Methods:Fifty chemotherapy-naive patients received nab-paclitaxel 125 mg/m2 plus gemcitabine 1,000 mg/m2 (days 1, 8, and 15) and nivolumab 3 mg/kg (days 1 and 15) in 28-day cycles. The primary endpoints were dose-limiting toxicities (DLTs; part 1) and grade 3/4 treatment-emergent adverse events (TEAEs) or treatment discontinuation due to TEAEs (parts 1/2). Secondary efficacy endpoints were progression-free survival (PFS), overall survival (OS), and response. Assessment of programmed cell death-ligand 1 (PD-L1) expression was an exploratory endpoint; additional biomarkers were assessed post hoc.Results:One DLT (hepatitis) was reported in part 1 among six DLT-evaluable patients; 48 of 50 patients experienced grade 3/4 TEAEs and 18 discontinued treatment due to TEAEs. One grade 5 TEAE (respiratory failure) was reported. Median [95% confidence interval (CI)] PFS/OS was 5.5 (3.25–7.20 months)/9.9 (6.74–12.16 months) months, respectively [median follow-up for OS, 13.6 months (95% CI, 12.06–23.49 months)]. Overall response rate (95% CI) was 18% (8.6%–31.4%). Median PFS/OS was 5.5/9.7 months (PD-L1 + CD8+/CD4+ cells increased significantly from baseline to cycle 3; median peak on-treatment Ki67+ CD8+ T-cell values were higher in responders than in nonresponders.Conclusions:The safety profile of nivolumab plus nab-paclitaxel and gemcitabine at standard doses in advanced pancreatic cancer was manageable, with no unexpected safety signals. Overall, the clinical results of this study do not support further investigation.

Percentage lymphocyte subset Ki67+. All patient visits are binned by treatment cycle. Data at baseline and treatment cycle 3 were compared using the Wilcoxon signed rank test. All available data are illustrated; however, statistical comparisons were performed only for data available at both baseline and treatment cycle 3 (n=29).

T-Cell Tumor Infiltration in Tissue-Matched Baseline (screening) and On-Treatment Metastatic Tumors (arm B parts 1 and 2)

Investigator-assessed PFS and OS in patients with PD-L1 expression cutoff of 1% (A, B) or 5% (C, D). HR, hazard ratio; mo, months; OS, overall survival; PD-L1, programmed cell death ligand-1; PFS, progression-free survival.

Patients were enrolled in Arm B Part 1 after the safety oversight committee deemed Arm A Part 1 safe. DRC, data review committee; LAPC, locally advanced pancreatic cancer; MPC, metastatic pancreatic cancer; RP2D, recommended Part 2 dose.

Concentrations of CXCL10 (A) and sIL2Rα (B) at baseline and peak on-treatment values. Investigator-assessed PFS in patients with high or low serum cytokines CXCL10 (C) and sIL2Rα (D). CXCL10, interferon gamma-induced protein 10; mo, months; NE, not estimable; NR, nonresponder; PFS, progression-free survival; R, responder; sIL2Rα, soluble form of interleukin 2 receptor alpha; Tx, treatment.

Data Tables

A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 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. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).

Purpose: Assess safety and efficacy of nivolumab plus nab-paclitaxel and gemcitabine in patients with locally advanced/metastatic pancreatic cancer in a two-part, open-label, phase I trial. Patients and Methods: Fifty chemotherapy-naive patients received nab-paclitaxel 125 mg/m2 plus gemcitabine 1,000 mg/m2 (days 1, 8, and 15) and nivolumab 3 mg/kg (days 1 and 15) in 28-day cycles. The primary endpoints were dose-limiting toxicities (DLTs; part 1) and grade 3/4 treatment-emergent adverse events (TEAEs) or treatment discontinuation due to TEAEs (parts 1/2). Secondary efficacy endpoints were progression-free survival (PFS), overall survival (OS), and response. Assessment of programmed cell death-ligand 1 (PD-L1) expression was an exploratory endpoint; additional biomarkers were assessed post hoc. Results: One DLT (hepatitis) was reported in part 1 among six DLT-evaluable patients; 48 of 50 patients experienced grade 3/4 TEAEs and 18 discontinued treatment due to TEAEs. One grade 5 TEAE (respiratory failure) was reported. Median [95% confidence interval (CI)] PFS/OS was 5.5 (3.25–7.20 months)/9.9 (6.74–12.16 months) months, respectively [median follow-up for OS, 13.6 months (95% CI, 12.06–23.49 months)]. Overall response rate (95% CI) was 18% (8.6%–31.4%). Median PFS/OS was 5.5/9.7 months (PD-L1 Conclusions: The safety profile of nivolumab plus nab-paclitaxel and gemcitabine at standard doses in advanced pancreatic cancer was manageable, with no unexpected safety signals. Overall, the clinical results of this study do not support further investigation.

Avadomide is a cereblon E3 ligase modulator and a potent antitumor and immunomodulatory agent. Avadomide trials are challenged by neutropenia as a major adverse event and a dose-limiting toxicity. Intermittent dosing schedules supported by preclinical data provide a strategy to reduce frequency and severity of neutropenia; however, the identification of optimal dosing schedules remains a clinical challenge. Quantitative systems pharmacology (QSP) modeling offers opportunities for virtual screening of efficacy and toxicity levels produced by alternative dose and schedule regimens, thereby supporting decision-making in translational drug development. We formulated a QSP model to capture the mechanism of avadomide-induced neutropenia, which involves cereblon-mediated degradation of transcription factor Ikaros, resulting in a maturation block of the neutrophil lineage. The neutropenia model was integrated with avadomide-specific pharmacokinetic and pharmacodynamic models to capture dose-dependent effects. Additionally, we generated a disease-specific virtual patient population to represent the variability in patient characteristics and response to treatment observed for a diffuse large B-cell lymphoma trial cohort. Model utility was demonstrated by simulating the avadomide effect in the virtual population for various dosing schedules and determining the incidence of high-grade neutropenia, its duration, and the probability of recovery to low-grade neutropenia.

Avadomide is a cereblon E3 ligase modulator and a potent antitumor and immunomodulatory agent. Avadomide trials are challenged by neutropenia as a major adverse event and a dose-limiting toxicity. Intermittent dosing schedules supported by preclinical data provide a strategy to reduce frequency and severity of neutropenia; however, the identification of optimal dosing schedules remains a clinical challenge. Quantitative systems pharmacology (QSP) modeling offers opportunities for virtual screening of efficacy and toxicity levels produced by alternative dose and schedule regimens, thereby supporting decision-making in translational drug development. We formulated a QSP model to capture the mechanism of avadomide-induced neutropenia, which involves cereblon-mediated degradation of transcription factor Ikaros, resulting in a maturation block of the neutrophil lineage. The neutropenia model was integrated with avadomide-specific pharmacokinetic and pharmacodynamic models to capture dose-dependent effects. Additionally, we generated a disease-specific virtual patient population to represent the variability in patient characteristics and response to treatment observed for a diffuse large B-cell lymphoma trial cohort. Model utility was demonstrated by simulating the avadomide effect in the virtual population for various dosing schedules and determining the incidence of high-grade neutropenia, its duration, and the probability of recovery to low-grade neutropenia. Supplementary Information The online version contains supplementary material available at 10.1208/s12248-021-00623-8.

CC-99282 is a novel CELMoD® agent that was optimized for activity in non-Hodgkin lymphoma (NHL). CC-99282 potently degrades Ikaros and Aiolos, resulting in enhanced antiproliferative, apoptotic, and immune-stimulatory activity in diffuse large B-cell lymphoma (DLBCL) models, including those with acquired chemoresistance (Lopez-Girona A, et al. Hematol Oncol. 2021). In lymphocytes, Ikaros negatively regulates gene expression via histone modifications, including polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 trimethylation (H3K27me3) (Oravecz A, et al. Nat Commun. 2015); in NHL, these epigenetic mechanisms are unclear. Using DLBCL models, we explored the relationship between CC-99282 activity and epigenetic status, and the mechanism of synergy of CC-99282 treatment and inhibition of EZH2, a PRC2 component. Baseline characteristics of human DLBCL lines and effects of CC-99282 ± tazemetostat (TAZ), a representative EZH2i, were evaluated in vitro and in vivo by chromatin immunoprecipitation sequencing, gene expression, flow cytometry, immunoblotting, enzyme fragment complementation assays, and/or CRISPR/Cas9 gene editing. Analysis of Ikaros/Aiolos degradation in > 20 DLBCL cell lines showed their loss is necessary, but not sufficient for CC-99282 efficacy. Evaluation of baseline histone marks showed that the sensitive cell lines exhibit aberrant, higher H3K27me3 coverage at promoter regions of expressed genes. This suggests a direct correlation between H3K27me3 status at these regions and CC-99282 sensitivity. In T cells, Oravecz et al. found that loss of Ikaros reduces H3K27me3 at specific chromatin sites due to its interaction with PRC2. We confirmed that these affected regions are enriched in genes upregulated upon CC-99282 treatment in DLBCL cell lines, suggesting a similar role for Ikaros in DLBCL. Pathway analysis following CC-99282 or TAZ treatment demonstrated high overlap between pathways altered by both agents. CC-99282 + TAZ combined demonstrated additive and/or synergistic antiproliferative and apoptotic effects in DLBCL cell lines. This combination did not alter Ikaros degradation or overall H3K27me3 status but increased downstream effects. Results were confirmed by CRISPR/Cas9 knockout competition assays with EZH2 and other PRC2 components. This effect was independent of cell of origin, EZH2 mutational status, or degree of CC-99282 sensitivity. Synergy was confirmed using the SU-DHL6 and DB xenograft models that are intrinsically resistant to EZH2is and CC-99282, respectively. Combination treatment yielded durable responses and tumor-free animals. Collectively, these data suggest that Ikaros could act as an epigenetic modulator through PRC2 recruitment and support the combination of CC-99282 with EZH2is in NHL (NCT03930953) to favor broad and durable clinical responses. Citation Format: Soraya Carrancio, Celia Fontanillo, Ryan Galasso, Martino Colombo, Scott Wood, Carla Guarinos, Alejandro Panjkovich, Diana Jankeel, Adam Blattler, Preethi Janardhanan, Matthew Groza, Jim Leisten, Rama Krishna Narla, Antonia Lopez-Girona, Daniel W. Pierce. Pathway interaction and mechanistic synergy of CC-99282, a novel cereblon E3 ligase modulator (CELMoD) agent, with enhancer of zeste homolog 2 inhibitors (EZH2is) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3932.

Assess safety and efficacy of nivolumab plusFifty chemotherapy-naive patients receivedOne DLT (hepatitis) was reported in part 1 among six DLT-evaluable patients; 48 of 50 patients experienced grade 3/4 TEAEs and 18 discontinued treatment due to TEAEs. One grade 5 TEAE (respiratory failure) was reported. Median [95% confidence interval (CI)] PFS/OS was 5.5 (3.25-7.20 months)/9.9 (6.74-12.16 months) months, respectively [median follow-up for OS, 13.6 months (95% CI, 12.06-23.49 months)]. Overall response rate (95% CI) was 18% (8.6%-31.4%). Median PFS/OS was 5.5/9.7 months (PD-L15%) and 6.8/11.6 months (PD-L1 ≥5%), respectively. Proportion of peripheral Ki67The safety profile of nivolumab plus

CC-99282 is a novel, oral CELMoD ® agent currently under investigation in phase 1 clinical studies in patients with relapsed or refractory (R/R) non-Hodgkin lymphomas (NHL) and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Mechanistically, CC-99282 interacts with the CRL4 CRBN E3 ubiquitin ligase substrate receptor CRBN to induce recruitment and ubiquitin-mediated proteasomal degradation of transcription factors Ikaros and Aiolos. The design intent for CC-99282 included efficient absorption, deep tissue distribution, and prolonged exposure to optimize activity in bulky lymphoma lesions. Recently, we reported that CC-99282 shows potent antitumor activity in different preclinical models of diffuse large B cell lymphoma (DLBCL; Lopez-Girona, et al. Hematol Oncol. 2021). Here, we provide an expanded analysis of CC-99282 activity as a monotherapy, as well as examine its synergistic activity with anti-CD20 antibody treatment, in preclinical models of NHL including DLBCL and follicular lymphoma (FL). Compared with existing agents targeting Ikaros/Aiolos that show activity in hematologic malignancies, such as lenalidomide, avadomide, and iberdomide (CC-220), CC-99282 induced a more rapid, deep, and sustained degradation of Ikaros/Aiolos, causing derepression of cyclin-dependent kinase (CDK) inhibitors and interferon-stimulated genes (IRF7, IFIT3, and DDX58), and the reduction of the highly critical oncogenic factors c-Myc and IRF4. These molecular changes were followed by potent, 10- to 100-fold enhanced, autonomous cell killing and induction of apoptosis (Figure). Our results show that these effects were independent of the cell of origin (activated B cell [ABC; TMD8 cell line], germinal center B cell [GCB; WSU-DLCL2 cell line], or primary mediastinal B cell lymphoma [PMBL] subtypes of DLBCL) or presence of high-risk chromosomal translocations (MYC, BCL2, and/or BCL6), as observed in a panel of 36 lymphoma cell lines that included DLBCL and FL cell lines. In vivo, CC-99282 demonstrated robust tissue distribution that favored target tissues and exhibited antitumor activity resulting in improved tumor regression and tumor-free animals in several lymphoma xenograft models, including an intracranial xenograft model. This strong antitumor activity was observed using various continuous and intermittent dosing paradigms. The potent, direct autonomous cell-killing activity of CC-99282 was augmented when CC-99282 was combined with the anti-CD20 antibody rituximab. In vitro combination studies of CC-99282 with rituximab in lymphoma cell lines demonstrated enhanced cell killing by human natural killer (NK) cells, macrophage-mediated phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In FL and DLBCL cell lines, we showed that the combination of CC-99282 with rituximab resulted in increases in both NK-mediated ADCC and macrophage-mediated ADCP of up to 20% compared with rituximab treatment alone. In vivo, combination treatment with CC-99282 and rituximab induced dose-dependent tumor growth inhibition in WSU-DLCL2 and RL (FL) xenograft models. In the WSU-DLCL2 model, CC-99282 (1 mg/kg) or rituximab (10 mg/kg) monotherapy resulted in modest tumor growth inhibition, whereas the combination of CC-99282 (1 mg/kg) and rituximab (10 mg/kg) resulted in tumor regression in 100% of animals. Similar results were obtained in FL xenograft models using the RL cell line, where combinations of CC-99282 (1 mg/kg) with rituximab (25 mg/kg) induced complete tumor regression in 100% of animals. In conclusion, CC-99282 is a novel CELMoD agent with an improved substrate degradation profile compared with existing Ikaros/Aiolos-degrading agents. CC-99282 demonstrated enhanced antiproliferative and apoptotic activities across a broad range of lymphoma cells and a robust distribution profile that favors target tissues such as lymphoid organs. In addition, CC-99282 acts synergistically in combination with anti-CD20 monoclonal antibody treatment. Collectively, these data support the clinical investigation of CC-99282 as monotherapy and in combination with rituximab in patients with R/R NHL. Figure 1 Figure 1. Disclosures Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Groocock: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Janardhanan: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jankeel: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Galasso: Ryan Galasso: Current Employment, Current equity holder in publicly-traded company. Guarinos: Bristol Myers Squibb: Current Employment. Narla: Bristol Myers Squibb: Current Employment. Groza: Bristol Myers Squibb: Current Employment. Leisten: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Rolfe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lopez-Girona: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Introduction: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD ®) agent that is a first-in-class degrader of translation termination factor G1 to S phase transition 1 (GSPT1). CC-90009 has demonstrated antileukemic activity as a single agent and is currently under investigation in patients with acute myeloid leukemia (AML; NCT02848001). Treatment with CC-90009 led to rapid reductions in peripheral and bone marrow blasts, and demonstrated preliminary promising efficacy, including several complete remissions, in patients with relapsed or refractory AML. Here, we describe the identification and preclinical activity of select anti-AML agents as potential combination partners of CC-90009 to further improve its efficacy and therapeutic index. Based on these results, the combination activity of CC-90009 with venetoclax (VEN)/azacitidine (AZA) is being evaluated in a phase 1/2 trial in patients with AML (NCT04336982). Methods: A high-throughput cell viability screen was performed to identify synergistic partners of CC-90009. AML cell lines (HL-60, HNT-34, KG-1, ML-2, NOMO-1, MOLM-13, MV4-11, F-36P, OCI-AML2, OCI-AML3) were treated with CC-90009 in combination with > 70 compounds, including standard anti-AML agents, tyrosine kinase inhibitors (TKIs), unfolded protein response inducers, transcription inhibitors, and epigenetic agents. MOLM-13 and MV4-11 are fms-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) cell lines. Hits were validated in a colony formation (CF) assay using primary AML cells and bone marrow mononuclear cells (BMMC) from healthy donors. Synergy of the combination partners with CC-90009 was further assessed in AML patient-derived xenograft (PDX) models. Synergy between the isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib and CC-90009 was evaluated in a TF-1 erythroleukemia cell line overexpressing IDH2 R140Q mutant, and an IDH2 R140Q PDX model, AM7577. Results: Our high-throughput combination screen revealed multiple TKIs, epigenetic agents, and pro-apoptotic agents as potential combination partners of CC-90009 in AML cell lines. FLT3 inhibitors, including sunitinib, pexidartinib, midostaurin, lestaurtinib, crenolanib, and gilteritinib, synergized with CC-90009 to reduce viability in FLT3-ITD AML cell lines MV4-11 and MOLM-13. Similarly, the B-cell lymphoma 2 (BCL2) inhibitor VEN potentiated CC-90009-induced apoptosis and accelerated cell-autonomous killing. Reduction in levels of MCL-1, an anti-apoptotic factor, by CC-90009 most likely contributed to the synergy with VEN. We prioritized the evaluation of FLT3, BCL2, and IDH2 inhibitors as partners of CC-90009. In CF assays, midostaurin enhanced the inhibitory effect of CC-90009 in primary AML cells, without augmenting the effect of CC-90009 in CD34+ BMMC from healthy donors. Similarly, VEN enhanced the reduction in CF by CC-90009 in AML patient-derived BMMC without exacerbating the decrease in CF by CC-90009 in BMMC from healthy donors. We characterized FLT3 inhibitor/CC-90009 and BCL2 inhibitor/AZA/CC-90009 combinations in a FLT3-ITD PDX murine model, PDX1. The FLT3 inhibitor quizartinib significantly prolonged survival when combined with CC-90009 compared with either agent alone (P < 0.001). Similarly, VEN/AZA/CC-90009 combination markedly extended survival compared with single agents or VEN/AZA doublets (P < 0.001). The synergy between CC-90009 and epigenetic modulators was validated and further characterized in customized cell differentiation assays. Addition of CC-90009 to enasidenib, a mutant IDH2 inhibitor, enhanced differentiation and killing of CD34+ stem and progenitor cells, and increased differentiated CD235a+ erythroblasts, compared with enasidenib alone in a TF-1 cell line overexpressing IDH2 R140Q. Enasidenib/CC-90009 combination treatment reduced CD45+ malignant populations and increased differentiated CD14+ cells, leading to significantly prolonged animal survival in an IDH2 R140Q PDX model, AM7577, compared with either agent alone (P < 0.0001). Conclusion: Using a high-throughput combination screen, we identified rational combination partners that synergize with CC-90009 in in vitro and in vivo AML models. Collectively, these results support the clinical evaluation of CC-90009 in combination with FLT3, BCL2, and IDH2 inhibitors to further improve treatment outcomes for patients with AML. Disclosures Pierce: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Yao: Bristol Myers Squibb: Consultancy, Current equity holder in publicly-traded company, Ended employment in the past 24 months, Research Funding. Pace: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Wang: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Flandin-Blety: Bristol Myers Squibb: Current Employment. Benitez: Bristol Myers Squibb: Current Employment. Guarinos: Bristol Myers Squibb: Current Employment. Hoffmann: Bristol Myers Squibb: Current Employment. Carrancio: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Pourdehnad: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties.

Introduction: CC-92480 is an oral novel cereblon (CRBN) E3 ligase modulator (CELMoD) agent currently in phase 1/2 development in patients with relapsed/refractory multiple myeloma (RRMM). CC-92480 induces rapid degradation of the transcription factors Ikaros and Aiolos, leading to apoptosis of myeloma cells and immune-stimulatory effects. The innovative CC-92480-MM-001 phase 1 study design investigated the effects of a broad range of CC-92480 doses and schedules. The pharmacodynamic effects will be discussed in this analysis with a focus on modulation of immune cell subsets. Methods: An adaptive Bayesian dose-escalation design was used in the CC-92480-MM-001 study (NCT03374085) that allowed for evaluation of several dosing schedules. The continuous schedules included 10 days on followed by 4 days off × 2, and 21 days on followed by 7 days off in a 28-day cycle. The intensive schedules included 3 days on followed by 11 days off × 2, and 7 days on followed by 7 days off × 2. An intensive biomarker sampling program was included in the study to allow for characterization of pharmacodynamic changes. Peripheral blood and bone marrow aspirates (BMA) were obtained from patients enrolled in part 1 of the dose-escalation study for biomarker analysis. Samples were taken pre-dose and during treatment at multiple time points. Biomarker analyses included Ikaros and Aiolos levels in peripheral blood mononuclear cells by flow cytometry; CRBN, Aiolos, Ikaros, and ZFP91 expression by immunohistochemistry (IHC) in BMA; weekly levels of serum free light chain (sFLC) and soluble B-cell maturation antigen (sBCMA), and effects on immune cells in peripheral blood. CC-92480 plasma exposures were collected at several time points in Cycle 1. Results: CC-92480 1.0 mg, 21/28-days was selected as the recommended phase 2 dose (RP2D) based on tolerability, efficacy, and pharmacodynamic effects. Degradation of Ikaros and Aiolos was evident at all dose levels in bone marrow plasma cells independent of baseline CRBN staining intensity and prior treatment. In these heavily pretreated patients with RRMM, CC-92480 induced rapid and sustained decreases in sFLC (median 94%) and sBCMA (median 78%) in Cycle 1 at the RP2D. Ikaros and Aiolos degradation in peripheral blood T cells was dose-dependent and reached > 80% degradation at 0.6 mg and above. Substrate recovery occurred during drug holidays, with faster recovery at lower doses, and reached full recovery with ≥ 7-day breaks. Focusing on the immunomodulatory effects, dynamic and dose- and schedule-dependent changes were demonstrated in immune cell subtypes. B cells decreased with increasing dose and reached > 90% at 0.8 mg and above in the continuous schedules. Recovery was evident during drug holidays, especially at lower doses. T-cell proliferation by Ki-67 staining increased between 30% and 350% during treatment and returned toward baseline during drug holidays at lower doses and with longer breaks. NK-cell proliferation by Ki-67 staining was evident at all doses and peaked at approximately 1 week post dose regardless of schedule. T cells demonstrated a shift from naïve to effector phenotype at all doses and schedules, and showed an increase in activation markers, HLA-DR, CD38, and ICOS. Regulatory T cells increased by 90% and 130% at 0.8 mg and 1.0 mg, respectively. At the 1.0 mg dose (RP2D) and more continuous schedules (10/14 days × 2 and 21/28 days), higher percentages of proliferating CD3+CD4+ and CD3+CD8+ T cells associated with a clinical response. Conclusions: The novel study design and biomarker sampling have allowed us to understand how depth, duration, and recovery of Ikaros and Aiolos degradation lead to specific immune changes. CC-92480 pharmacodynamic activity was dose-dependent from 0.1 mg to 1.0 mg and recovery was dose- and schedule-dependent. These changes were also demonstrated in immune cell subsets. Dynamic changes of immune cell subsets in peripheral blood occurred in concert with Ikaros and Aiolos degradation and recovery, suggesting that immune profiles may be modified and optimized by dose and schedule. This new insight may be utilized to provide the rationale for dose and schedule for specific immune effects when combining with other immunotherapies, such as bispecific antibodies and CAR T cells. Disclosures Wong: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Lamba:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company; Pfizer: Current equity holder in publicly-traded company. Jiménez Nuñez:CITRE, a Bristol-Myers Squibb Company, Spain: Current Employment. Bauer:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Richardson:Celgene/BMS, Oncopeptides, Takeda, Karyopharm: Research Funding. Bahlis:Sanofi: Consultancy, Honoraria; BMS/Celgene and Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Research Funding; Takeda: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Karyopharm Therapeutics: Consultancy, Honoraria; GSK: Consultancy, Honoraria; Genentech: Consultancy, Honoraria. Ramasamy:Takeda: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Oncopeptides: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Trudel:Pfizer: Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Genentech: Research Funding; GSK: Consultancy, Honoraria, Research Funding; Takeda, Karyopharm, AstraZeneca, Sanofi: Honoraria; Janssen: Honoraria, Research Funding; Amgen: Consultancy, Research Funding. Martínez-Lopez:Altum, Hosea: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Vivia Biotech: Honoraria; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Roche: Speakers Bureau; Janssen: Speakers Bureau; Incyte: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding. Mateos:EDOMundipharma: Consultancy; Adaptive: Consultancy; Pharmamar: Consultancy; GlaxoSmithKline: Consultancy; AbbVie: Consultancy; Takeda: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Janssen: Consultancy. Rodriguez-Otero:Oncopeptides: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Medscape: Membership on an entity's Board of Directors or advisory committees; Celgene/Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Sanofi: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Current Employment, Current equity holder in publicly-traded company, Honoraria; Kite: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria. Lonial:TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Other: Personal fees, Research Funding; Merck: Consultancy, Honoraria, Other: Personal fees; GSK: Consultancy, Honoraria, Other: Personal fees; Abbvie: Consultancy; Takeda: Consultancy, Other: Personal fees, Research Funding; Genentech: Consultancy; Karyopharm: Consultancy; Sanofi: Consultancy; Janssen: Consultancy, Honoraria, Other: Personal fees, Research Funding; JUNO Therapeutics: Consultancy; Millennium: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Other: Personal fees; Amgen: Consultancy, Honoraria, Other: Personal fees; Onyx: Honoraria. Popat:AbbVie: Consultancy, Honoraria; GSK: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Janssen: Consultancy, Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Takeda: Consultancy, Honoraria, Other: Travel support, Research Funding; Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Oriol:Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Orlowski:Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees; Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties; Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; STATinMED Research: Consultancy; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding. Berdeja:Amgen: Consultancy, Research Funding; Abbvie: Research Funding; Bluebird: Research Funding; Servier: Consultancy; Takeda: Consultancy, Research Funding; Vivolux: Research Funding; Kesios: Research Funding; Karyopharm: Consultancy; Glenmark: Research Funding; Genentech, Inc.: Research Funding; EMD Sorono: Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Cellularity: Research Funding; Constellation: Research Funding; CRISPR Therapeutics: Consultancy, Research Funding; CURIS: Research Funding; Poseida: Research Funding; Prothena: Consultancy; Kite Pharma: Consultancy; Legend: Consultancy; Lilly: Research Funding; Novartis: Research Funding; Teva: Research Funding; Acetylon: Research Funding; Bioclinica: Consultancy; BMS: Consultancy, Research Funding. Pourdehnad:Celgene: Ended employment in the past 24 months, Patents & Royalties: Various CC-122 patents; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: Various CC-122 patents. Pierce:Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

Background: Albumin-bound paclitaxel (nab-paclitaxel, nab-PTX) plus gemcitabine (GEM) combination has demonstrated efficient antitumour activity and statistically significant overall survival of patients with metastatic pancreatic ductal adenocarcinoma (PDAC) compared with GEM monotherapy. This regimen is currently approved as a standard of care treatment option for patients with metastatic PDAC. It is unclear whether cremophor-based PTX combined with GEM provide a similar level of therapeutic efficacy in PDAC. Methods: We comprehensively explored the antitumour efficacy, effect on metastatic dissemination, tumour stroma and survival advantage following GEM, PTX and nab-PTX as monotherapy or in combination with GEM in a locally advanced, and a highly metastatic orthotopic model of human PDAC. Results: Nab-PTX treatment resulted in significantly higher paclitaxel tumour plasma ratio (1.98-fold), robust stromal depletion, antitumour efficacy (3.79-fold) and survival benefit compared with PTX treatment. PTX plus GEM treatment showed no survival gain over GEM monotherapy. However, nab-PTX in combination with GEM decreased primary tumour burden, metastatic dissemination and significantly increased median survival of animals compared with either agents alone. These therapeutic effects were accompanied by depletion of dense fibrotic tumour stroma and decreased proliferation of carcinoma cells. Notably, nab-PTX monotherapy was equivalent to nab-PTX plus GEM in providing survival advantage to mice in a highly aggressive metastatic PDAC model, indicating that nab-PTX could potentially stop the progression of late-stage pancreatic cancer. Conclusions: Our data confirmed that therapeutic efficacy of PTX and nab-PTX vary widely, and the contention that these agents elicit similar antitumour response was not supported. The addition of PTX to GEM showed no survival advantage, concluding that a clinical combination of PTX and GEM may unlikely to provide significant survival advantage over GEM monotherapy and may not be a viable alternative to the current standard-of-care nab-PTX plus GEM regimen for the treatment of PDAC patients.

8531 Background: CC-92480 is a novel cereblon (CRBN) E3 ligase modulator (CELMoD) agent under investigation in a first-in-human phase 1 study (NCT03374085) in RRMM patients (pts). In preclinical studies, CC-92480 demonstrated efficient and sustained degradation of Ikaros/Aiolos leading to broad antiproliferative effects and induction of apoptosis in MM cell lines, and enhanced immune stimulatory effects. Methods: Eligible RRMM pts received escalating doses of CC-92480 + dexamethasone. Several dosing schedules were evaluated in parallel; more continuous with 4-day or 7-day breaks and intensive with longer breaks in a 28-day cycle. Peripheral blood and bone marrow aspirates (BMA) were taken before and during treatment at multiple time points. Levels of Ikaros/Aiolos in T cells, and effects on immunomodulation were assessed by flow cytometry. Weekly levels of free light chain (sFLC) and B-cell maturation antigen (sBCMA) were determined in serum during the first 2 cycles of treatment. BMA clots were analyzed by immunohistochemistry for CRBN, Ikaros, Aiolos, ZFP91, c-Myc, and IRF-4. Results: The rate and depth of Ikaros/Aiolos degradation in T cells increased with dose and reached maximal at ≥0.6 mg QD with sustained degradation over 24 hrs. Substrate recovery occurred during drug holidays with faster recovery at lower doses, and reached full recovery with ≥7-day break for all dose levels tested. B cells decreased with increasing dose, and T-cell proliferation was demonstrated at all doses/schedules. Substrate degradation was also evident in bone marrow plasma cells including in the setting of low CRBN levels. In these heavily pretreated, including triple-class-refractory, RRMM pts, CC-92480 dosing periods led to rapid and sustained decreases in sFLC and sBCMA. This was dose and schedule dependent and correlated with plasma exposure; the longer breaks in the intensive schedules led to rapid rebound of these markers, while the more continuous schedules maintained the depth of suppression. Conclusions: PD responses correlated with dose and schedule. PD samplings at multiple time points during treatment allowed dynamic changes and kinetics of each biomarker in all schedules to be followed and to inform next steps. Ikaros/Aiolos degradation and recovery, coupled with changes in sFLC and sBCMA, guided the adjustment of the dosing schedule during dose escalation in order to optimize efficacy and tolerability. The study is ongoing and selection of the recommended phase 2 dose is pending.

8500 Background: CC-92480 is a novel cereblon E3 ligase modulator (CELMoD) agent designed for rapid, maximal degradation of Ikaros and Aiolos. In vitro, it has enhanced antiproliferative and tumoricidal activity in MM cell lines, including those resistant to lenalidomide (LEN) and pomalidomide (POM), with strong immune stimulatory activity. Methods: A phase 1, multicenter, dose-escalation study evaluated the maximum tolerated dose (MTD), recommended phase 2 dose, safety, tolerability, and pharmacokinetics of CC-92480 + DEX in heavily pretreated RRMM pts. Eligible pts had progression on or within 60 days of their last MM therapy and were either resistant or intolerant to, or not otherwise candidates for currently available therapies. Several treatment schedules tested escalating doses of CC-92480 + DEX (40 mg; 20 mg if ≥75 yrs). Results: As of Dec 24, 2019, 66 pts had received CC-92480 + DEX. Median age was 67 yrs (range 40–78), median number of prior regimens was 6 (range 2–13). Prior therapies included stem cell transplantation (67%), bortezomib (92%), LEN (89%), POM (83%), and anti-CD38 antibodies (78%). CC-92480 doses explored included 0.1–1.0 mg QD (10/14 days × 2), 0.8–1.0 mg QD (21/28 days), 0.2–0.8 mg BID (3/14 days × 2), and 1.6–2.0 mg QD (7/14 days × 2). MTD was 1.0 mg for both 10/14 × 2 and 21/28 schedules. Grade 3–4 treatment-emergent adverse events (TEAEs) were reported in 58 (88%) pts. Most frequent grade 3–4 TEAEs included neutropenia (53%), infections (30%), anemia (29%), and thrombocytopenia (17%), with 9% grade 3 fatigue. Among different cohorts,10 pts had dose-limiting toxicities (the majority related to neutropenia). Overall response rate (ORR) was 21% (9 very good partial responses [VGPRs]; 5 PRs) for efficacy evaluable population (n = 66). Efficacy was dose and schedule dependent; across two 1.0 mg QD schedules (10/14 × 2 and 21/28), 10 of 21 (48%) pts responded (7 VGPR and 3 PR), with response independent of immunomodulatory drug (IMiD) refractoriness. Plasma exposure increase and peripheral blood Ikaros and Aiolos degradation were dose dependent. Ikaros and Aiolos significantly decreased in bone marrow plasma cells of LEN- and POM-refractory pts. Conclusions: TEAEs of CC-92480 were mainly related to myelosuppression in heavily pretreated, including triple-class-refractory, RRMM pts. Promising activity with 48% ORR at therapeutic doses was observed. The study is ongoing to further optimize dose and schedule, with combination studies underway and dose expansion cohorts planned. Clinical trial information: NCT03374085 .

Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma that harbors tumor-promoting properties. No good biomarkers exist to monitor the effect of stromal targeting therapies or to predict response. We set out to identify such non-invasive markers for PDAC stroma and predict response to therapy. Gene expression datasets, co-culture experiments, xenografts, and patient samples were analyzed. Serum samples were measured from a cohort of 58 resected patients, and 87 metastatic or locally advanced PDAC patients. Baseline and follow-up levels were assessed in 372 additional metastatic PDAC patients who received nab-paclitaxel with gemcitabine (n = 184) or gemcitabine monotherapy (n = 188) in the phase III MPACT trial. Increased levels of ADAM12 were found in PDAC patients compared to healthy controls (p n = 157 and n = 38). High levels of ADAM12 significantly associated with poor outcome in resected PDAC (HR 2.07, p = 0.04). In the MPACT trial survival was significantly longer for patients who received nab-paclitaxel and had undetectable ADAM12 levels before treatment (OS 12.3 m vs 7.9 m p = 0.0046). Consistently undetectable or decreased ADAM12 levels during treatment significantly associated with longer survival as well (OS 14.4 m and 11.2 m, respectively vs 8.3, p = 0.0054). We conclude that ADAM12 is a blood-borne proxy for stromal activation, the levels of which have prognostic significance and correlate with treatment benefit.

Purpose: nab-Paclitaxel plus gemcitabine was superior to gemcitabine alone for patients with metastatic pancreatic cancer (MPC) in the phase III MPACT trial. This study evaluated the association of secreted protein acidic and rich in cysteine (SPARC) levels with efficacy as an exploratory endpoint. Experimental Design: Patients with previously untreated MPC (N = 861) received nab-paclitaxel plus gemcitabine or gemcitabine alone. Baseline SPARC level was measured in the tumor stroma and epithelia (archival biopsies) and plasma. Experiments were performed in pancreatic cancer mouse models in which SPARC was intact or deleted. Results: SPARC was measured in the tumor stroma of 256 patients (30%), the tumor epithelia of 301 patients (35%), and plasma of 343 patients (40%). Stroma-evaluable samples were from metastases (71%), from the pancreas (11%), or of unidentifiable origin (insufficient tissue to determine; 17%). For all patients, stromal SPARC level [high (n = 71) vs. low (n = 185)] was not associated with overall survival (OS; HR, 1.019; P = 0.903); multivariate analysis confirmed this lack of association. There was no association between stromal SPARC level and OS in either treatment arm. Neither tumor epithelial SPARC nor plasma SPARC was associated with OS. Results from a SPARC knockout mouse model treated with nab-paclitaxel plus gemcitabine revealed no correlation between SPARC expression and tumor progression or treatment efficacy. Conclusions: SPARC levels were not associated with efficacy in patients with MPC. This exploratory analysis does not support making treatment decisions regarding nab-paclitaxel plus gemcitabine or gemcitabine alone in MPC based on SPARC expression. Clin Cancer Res; 21(21); 4811–8. ©2015 AACR.

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.

Background: CC-90009 is a novel cereblon E3 ligase modulator (CELMoD), which is currently under investigation in a first-in-human, phase I study (CC-90009-AML-001; NCT02848001) in patients with R/R AML. In preclinical models, CC-90009 drives the binding of the target protein, translation termination factor G1 to S phase transition 1 (GSPT1), to cereblon and induces its ubiquitination and proteasome-dependent degradation. Loss of GSPT1 results in activation of the integrated stress response (ISR), inhibition of nonsense-mediated decay (NMD), and induction of apoptosis. Deep degradation of GSPT1, mediated by CC-90009, led to AML cell death in vitro and potent antitumor activity in patient-derived AML xenograft models. In the ongoing phase I study, CC-90009 has demonstrated antileukemic activity. Here, we characterize the pharmacodynamic responses using a suite of novel assays to support CC-90009 dose and schedule optimization. Methods: Adult patients with R/R AML received intravenous CC-90009 daily on Days 1-5 (D1-5 schedule) or on Days 1-3 and 8-10 (D1-3/8-10 schedule) of a 28-day cycle. Peripheral blood samples taken before, during, and after dosing in the first treatment cycle were analyzed. Levels of intracellular GSPT1 in blasts and normal blood cell types were quantitated by flow cytometry analysis. Transcript levels of ISR and NMD variants in peripheral blood mononuclear cells (PBMC) were measured by qPCR. Bone marrow (BM) core biopsies at screening, Cycle 1 Day 5 and 28, and Cycle 2 and 4 Day 28, were analyzed for GSPT1, cleaved caspase 3, and CD34 protein expression by immunohistochemistry. ATF3 and DDIT3 mRNA levels were assessed in BM samples by RNA in situ hybridization. Results: The rate and depth of GSPT1 loss in T cells and in circulating AML blasts increased with dose. A marked reduction in GSPT1 was observed in T cells and blast cells of most patients after the first dose of CC-90009 at all dose levels, and GSPT1 levels approached the assay floor between Days 2 and 5 at doses of 1.2 mg and higher on the D1-5 schedule. At 2.4 mg and higher on the D1-5 schedule, a reduction in GSPT1 levels of > 90% was observed in T cells (19 of 29 patients) and in blast cells (11 of 29 patients), with stronger GSPT1 reductions detected in AML blasts and normal T cells compared with B cells or granulocytes. In the 3 mg D1-5 cohort, patients with sustained GSPT1 reduction in peripheral blasts in the days following treatment had more persistent blast suppression compared with patients showing an earlier rebound of GSPT1. At 3 mg and 3.6 mg dose levels, continuous treatment (D1-5) resulted in slower kinetics of GSPT1 rebound and conferred superior antileukemic activity compared with the intermittent dosing schedule (D1-3/D8-10). In addition to measuring the direct target of CC-90009, GSPT1, we also investigated markers downstream of GSPT1 degradation. Several patients with deep and sustained GSPT1 loss in the high-dose cohorts (2.4 mg and above) showed increased levels of ISR-related transcripts (ATF3 and DDIT3) and NMD-associated splice variants (SRSF3 and SRSF6) in on-treatment PBMC samples. Similarly, in BM, deep GSPT1 loss coincided with induction of ATF3 and DDIT3 mRNA, increased cleaved caspase 3 expression, and reduced CD34+ blasts. These clinical findings are consistent with our preclinical studies in which GSPT1 loss culminated in apoptosis, which may be mediated through activation of ISR and inhibition of NMD pathways. Conclusions: CC-90009 is a novel CELMoD and a first-in-class GSPT1 degrader. A suite of novel pharmacodynamic assays performed on patient-derived peripheral blood cells and BM demonstrated a dose-dependent modulation of GSPT1, and showed that the preclinical mechanisms of ISR induction, NMD inhibition, and apoptosis can be confirmed in AML cells in patients. Deeper and more rapid GSPT1 degradation as well as delayed rebound were associated with more rapid, deeper, and more persistent blast reductions. Characterization of these pharmacodynamic responses in ongoing dose-schedule explorations will help identify the optimal scheme for the expansion phase and provide further insight into the mechanism of clinical response. Disclosures Fan: Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Yao:Celgene Corporation: Employment. Uy:Astellas: Consultancy; Pfizer: Consultancy; Curis: Consultancy; GlycoMimetics: Consultancy. Zeidan:Pfizer: Consultancy, Honoraria, Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding. Montesinos:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees. DeAngelo:Jazz Pharmaceuticals, Inc.: Consultancy; Takeda Pharmaceuticals: Consultancy; Shire: Consultancy; GlycoMimetics: Research Funding; Pfizer, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Incyte Corporation: Consultancy; Celgene Corporation: Consultancy; AbbVie, Inc.: Research Funding; Blue print Medicines: Consultancy, Research Funding; Amgen: Consultancy. Altman:Novartis: Consultancy; Cancer Expert Now: Consultancy; Biosight: Other: US Lead; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; France Foundation: Speakers Bureau; prIME Oncology: Speakers Bureau; PeerView: Speakers Bureau; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee. Koprivnikar:Amgen: Speakers Bureau; Abbvie: Speakers Bureau; Pfizer: Honoraria; Novartis: Speakers Bureau. Vyas:Astellas: Speakers Bureau; Pfizer: Speakers Bureau; Abbvie: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding. Fløisand:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Novartis: Honoraria. Gjertsen:Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; EU Horizon 2020: Research Funding; KinN Therapeutics AS: Equity Ownership; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees; ERA PerMed: Research Funding; The Norwegian Cancer Society: Research Funding; Helse Vest Health Trust: Research Funding; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Research Council of Norway: Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; ACTII AS: Equity Ownership. Buchholz:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership.

Background: CC-90009 is a cereblon (CRBN) E3 ligase modulator (CELMoD) and a first-in-class small molecule that drives the binding of a novel target protein, G1 to S phase transition 1 (GSPT1), to CRBN, resulting in the proteasome-dependent degradation of GSPT1. GSPT1 plays a central role in mRNA translation, and loss of GSPT1 activates an integrated stress response that leads to AML cell death (Matyskiela ME, et al. Nature. 2016;535:252-7; Zhouravleva G, et al. EBMO J. 1995;14:4065-72). In preclinical testing, CC-90009 is active across a range of AML cell lines and primary AML patient (pt) samples in vitro and in vivo and exerts its GSPT1- and CRBN-dependent effects through rapid induction of apoptosis. Here we share the first clinical results in pts with R/R AML. Methods: Adult pts with R/R AML enrolled in the dose-finding phase of this first-in-human, multicenter, open-label phase 1 study to evaluate tolerability, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacy of CC-90009; and to establish the recommended phase 2 dose and schedule (RP2D) (CC-90009-AML-001; NCT02848001). Dose escalation proceeded via a modified 3 + 3 design. Treatment was by daily intravenous administration on either Days 1-5 (D1-5) or Days 1-3 and 8-10 (D1-3/8-10) of a 28-day cycle. Treatment response was assessed after Cycles 1, 2, and 4 by modified International Working Group 2003 criteria. Safety and preliminary response data are presented for all treated pts. PK and PD were analyzed for evaluable pts. Results: As of May 15, 2019, 45 pts with R/R AML had been treated, including 35 pts on the D1-5 and 10 pts on the D1-3/D8-10 schedule. Median age was 66 years (range 27-81); 73% were male. Most pts (n = 36; 80%) were refractory to their last therapy and 17 pts (38%) were refractory to all prior therapy; 14 pts (31%) had secondary AML. Pts were treated at dose levels from 0.3 to 3.6 mg. Dose-limiting toxicities (DLTs) reported (only in dose levels from 2.4 to 3.6 mg) included hypotension, systemic inflammatory response syndrome (SIRS), hyperbilirubinemia, pneumonitis, and pericarditis with tamponade. Exploration of the 3.6 mg dose level is ongoing; the RP2D has not yet been determined. CC-90009-related grade 3/4 treatment-emergent adverse events (TEAEs) occurred in 23 pts (51%); those occurring in >1 pt were hypocalcemia (22%); hypotension (13%); and hyperbilirubinemia, hyperglycemia, hypophosphatemia, pneumonitis, sepsis, thrombocytopenia, and tumor lysis syndrome (4%). Preclinically identified hypocalcemia was confirmed as a CC-90009 on-target toxicity in the clinic; it was reversible, manageable and did not lead to any treatment discontinuations. The majority of treated pts experienced ≥1 serious TEAE (80%); most were infections (47%). Two (4%) pts experienced TEAEs leading to permanent discontinuation of the study drug. Dose interruptions due to TEAEs occurred in 12 pts (27%) and dose reductions in 2 pts (4%). Of 40 pts who discontinued treatment, 24 (60%) discontinued due to progressive disease or lack of efficacy. Seven pts discontinued treatment due to death; 4 deaths were secondary to progression from AML, 2 due to sepsis and 1 due to hyperglycemic hyperosmolar nonketotic syndrome. Responses to single-agent treatment were observed in pts treated at 3.0 or 3.6 mg on the D1-5 schedule, with a best response of complete remission (CR; n = 1), morphologic CR with incomplete blood count recovery (CRi; n = 1) and morphologic leukemia-free state (MLFS; n = 1). A dose-dependent decrease in GSPT1 levels in peripheral blood blasts and T cells was observed, with a >90% decrease observed for higher dose levels. Evidence of antileukemic activity (decreases in bone marrow and/or peripheral blasts) was seen in pts treated with CC-90009 at 1.2 mg and above with a trend to more sustained reductions at the highest dose levels. Plasma PK analysis demonstrated dose-dependent exposure. Conclusions: In this phase 1 study of CC-90009, a first-in-class agent, evidence of deep GSPT1 degradation, on-target activity and promising antileukemic activity was observed. The observed TEAEs, in addition to those expected in this heavily pretreated R/R AML pt population, were generally well manageable. The study is ongoing with further optimization of dose, schedule and toxicity mitigation. Expansion cohorts in R/R AML and higher-risk myelodysplastic syndromes are planned. Disclosures Uy: GlycoMimetics: Consultancy; Curis: Consultancy; Astellas: Consultancy; Pfizer: Consultancy. Montesinos:Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding. DeAngelo:Blue print Medicines: Consultancy, Research Funding; Celgene Corporation: Consultancy; Shire: Consultancy; Pfizer, Inc.: Consultancy; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Incyte Corporation: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; GlycoMimetics: Research Funding; AbbVie, Inc.: Research Funding; Takeda Pharmaceuticals: Consultancy; Amgen: Consultancy. Altman:Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Biosight: Other: US Lead; France Foundation: Speakers Bureau; PeerView: Speakers Bureau; Agios: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Consultancy, Honoraria, Other: Data Safety and Monitoring Committee; Cancer Expert Now: Consultancy; Theradex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; prIME Oncology: Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Koprivnikar:Amgen: Speakers Bureau; Pfizer: Honoraria; Abbvie: Speakers Bureau; Novartis: Speakers Bureau. Vyas:Astellas: Speakers Bureau; Abbvie: Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Forty Seven, Inc.: Research Funding; Daiichi Sankyo: Speakers Bureau; Pfizer: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Fløisand:Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; Novartis: Honoraria. Gjertsen:BerGenBio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; EU Horizon 2020: Research Funding; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; The Norwegian Cancer Society: Research Funding; KinN Therapeutics AS: Equity Ownership; ACTII AS: Equity Ownership; ERA PerMed: Research Funding; Helse Vest Health Trust: Research Funding; Research Council of Norway: Research Funding. Esteve:Astellas: Consultancy, Speakers Bureau; Amgen: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmaceuticals: Consultancy; Roche: Consultancy; Pfizer: Consultancy. Buchholz:Celgene Corporation: Employment, Equity Ownership. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Fan:Celgene Corporation: Employment, Equity Ownership. Hanna:Celgene Corporation: Employment, Equity Ownership. Li:Celgene Corporation: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership. Hege:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties; Mersana Therapuetics: Membership on an entity's Board of Directors or advisory committees; Society for Immunotherapy of Cancer: Membership on an entity's Board of Directors or advisory committees; Arcus Biosciences: Membership on an entity's Board of Directors or advisory committees. Pourdehnad:Celgene Corporation: Employment, Equity Ownership. Zeidan:Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Cardinal Health: Honoraria; Daiichi Sankyo: Honoraria; Novartis: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Astellas: Honoraria.

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.

Acute myeloid leukemia (AML) is a clonal malignant disease initiated and propagated by leukemia stem cells (LSCs). Both LSCs and normal hematopoietic stem cells (HSCs) share many biological properties including self-renewal and quiescence. One such shared property that we have recently established involves the pro-survival features of proteostatic stress signaling. Stem cells have reduced protein translation initiation due to scarcity of the eIF2α translation initiation complex (van Galen et al Nature 2014; Cell Reports 2018). This in turn, increases the activity of activating transcription factor 4 (ATF4) uniquely in HSCs and LSCs. In homeostasis, this level of ATF4 facilitates stem cell persistence and survival, but upon stronger stress activation stem cell apoptosis ensues. This mechanism predicts that agonists of the integrated stress response (ISR) could provide a novel therapeutic approach to eradicate LSCs. Here we report that the novel cereblon E3 ligase modulator (CELMoD) CC-90009, which causes degradation of the translation termination factor G1 to S phase transition protein 1 (GSPT1) and downstream activation of ISR, is potent against primary AML both in vitro and in vivo, and reduces self-renewing LSCs in preclinical xenograft models for human AML. We first carried out in vitro assays to evaluate the effect of CC-90009 on primary AML samples. We found that CC-90009 degraded GSPT1 in primary AML cells and induced leukemic cell apoptosis in 24 hours. Leukemic colony forming progenitors were also reduced by CC-90009 in a dose-dependent manner. We next tested the efficacy of CC-90009 against primary AML samples in xenografts in NOD/SCID mice. Leukemia cells were transplanted intrafemorally 21 days prior to CC-90009 treatment. Mice were treated with vehicle or CC-90009 at 2.5mg/kg BID for 4 weeks. Heterogeneous responses to the CC-90009 treatment were observed. Of 35 AML samples tested, 16 were highly responsive to CC-90009 with >75% reduction of AML engraftment, 10 showed moderate response between 45% and 75% reductions, and 9 showed reductions of To determine the efficacy of CC-90009 against AML cases at high risk of relapse following standard induction chemotherapy, we assessed CC-90009 efficacy vs. the status of an expression-based 17-gene leukemia stem cell score (the LSC17 score) that was recently implemented for rapid risk stratification of AML patients (Ng et al, Nature 2016). LSC17-high patients are predicted to have poor treatment response and poor clinical outcome. We found that, while 8 out of 9 poor responders to CC-90009 had high LSC17 scores, 20 out of 28 samples that had high LSC17 scores responded well to CC-90009, indicating that the drug is able to target high risk cases. Serial transplantation utilizing limiting dilution analysis showed that CC-90009 targeted self-renewing LSCs. Our data established that a new CELMoD CC-90009 has anti-proliferative effects on human primary AML cells and self-renewing LSCs evaluated in xenograft assays. These observations provide important implications for CC-90009 in its clinical development as a new therapeutic agent to treat AML patients with high risk disease when treated with standard of care therapies. Currently, a phase I study evaluating CC-90009 in relapsed or refractory AML is ongoing (CC-90009-AML-001; NCT02848001). Disclosures Jin: Trillium Therapeutics: Other: licensing agreement. Ng:Celgene: Research Funding. Wang:Pfizer AG Switzerland: Honoraria, Other: Travel and accommodation; Trilium therapeutics: Other: licensing agreement, Research Funding; NanoString: Other: Travel and accommodation; Pfizer International: Honoraria, Other: Travel and accommodation. Minden:Trillium Therapetuics: Other: licensing agreement. Fan:Celgene Corporation: Employment, Equity Ownership. Pierce:Celgene Corporation: Employment, Equity Ownership. Pourdehnad:Celgene Corporation: Employment, Equity Ownership.

B-cell receptor (BCR) signaling plays a key role in the pathogenesis of B-cell malignancies, mediating the survival and proliferation of malignant B cells.1,2 Clinical studies have shown that Bruton’s tyrosine kinase (BTK) inhibitors are well tolerated, with promising clinical activity. Ibrutinib has shown 30-month progression-free survival (PFS) of 69% in relapsed chronic lymphocytic leukemia (CLL) patients,3–5 and has substantial activity in mantle cell lymphoma and activated B-cell-type diffuse large B-cell lymphoma.6,7 CC-292 is a highly selective oral small-molecule inhibitor that binds covalently and irreversibly to the same cysteine 481 in BTK as ibrutinib, inhibiting its signaling.8 We report here the results of a phase I study of CC-292 in patients with relapsed/refractory (R/R) CLL/small lymphocytic lymphoma (SLL), B-cell non-Hodgkin lymphoma (B-NHL), and Waldenstrom macroglobulinemia (WM). A total of 113 patients received continuous dosing with CC-292 in 28-day cycles at doses ranging from 125 mg to 1000 mg once daily, and 375 mg and 500 mg twice daily, continuing into dose-expansion cohorts of 750 mg once daily and a preliminary recommended phase II dose (RP2D)-expansion cohort of 500 mg twice daily. Four patients experienced dose-limiting toxicity (DLT) but only one in any treatment cohort. The most frequent grade 3–4 adverse events (AEs) were neutropenia (16%) and thrombocytopenia (8%). The most common non-hematologic treatment-emergent AEs (TEAEs) of any grade were diarrhea (68%) and fatigue (45%). Twice-daily administration of CC-292 was instituted to improve sustained BTK occupancy, and, in fact, did result in more than 90% BTK receptor occupancy at both the 4- and 24-h post-dose time points. Efficacy in the CLL/SLL population (n=84) showed that overall response rate (ORR) in patients receiving twice-daily dosing was 53%; an additional 10% had partial response with lymphocytosis (PR-L). CC-292 was, therefore, well tolerated and achieved high nodal and PR rates in relapsed CLL/SLL patients, but showed less durability than other BTK inhibitors.

While advances in high-throughput screening have resulted in increased ability to identify synergistic anti-cancer drug combinations, validation of drug synergy in the in vivo setting and prioritization of combinations for clinical development remain low-throughput and resource intensive. Furthermore, there is currently no viable method for prospectively assessing drug synergy directly in human patients in order to potentially tailor therapies. To address these issues we have employed the previously described CIVO platform and developed a quantitative approach for investigating multiple combination hypotheses simultaneously in single living tumors. This platform provides a rapid, quantitative and cost effective approach to compare and prioritize drug combinations based on evidence of synergistic tumor cell killing in the live tumor context. Using a gemcitabine resistant model of pancreatic cancer, we efficiently investigated nine rationally selected Abraxane-based combinations employing only 19 xenografted mice. Among the drugs tested, the BCL2/BCLxL inhibitor ABT-263 was identified as the one agent that synergized with Abraxane® to enhance acute induction of localized apoptosis in this model of human pancreatic cancer. Importantly, results obtained with CIVO accurately predicted the outcome of systemic dosing studies in the same model where superior tumor regression induced by the Abraxane/ABT-263 combination was observed compared to that induced by either single agent. This supports expanded use of CIVO as an in vivo platform for expedited in vivo drug combination validation and sets the stage for performing toxicity-sparing drug combination studies directly in cancer patients with solid malignancies.

Decreased cardiac contractility is a central feature of systolic heart failure. Existing drugs increase cardiac contractility indirectly through signaling cascades but are limited by their mechanism-related adverse effects. To avoid these limitations, we previously developed omecamtiv mecarbil, a small-molecule, direct activator of cardiac myosin. Here, we show that it binds to the myosin catalytic domain and operates by an allosteric mechanism to increase the transition rate of myosin into the strongly actin-bound force-generating state. Paradoxically, it inhibits adenosine 5'-triphosphate turnover in the absence of actin, which suggests that it stabilizes an actin-bound conformation of myosin. In animal models, omecamtiv mecarbil increases cardiac function by increasing the duration of ejection without changing the rates of contraction. Cardiac myosin activation may provide a new therapeutic approach for systolic heart failure.

Kinesin is a dimeric motor protein that transports organelles in a stepwise manner toward the plus-end of microtubules by converting the energy of ATP hydrolysis into mechanical work. External forces can influence the behavior of kinesin, and force-velocity curves have shown that the motor will slow down and eventually stall under opposing loads of ≈5 pN. Using an in vitro motility assay in conjunction with a high-resolution optical trapping microscope, we have examined the behavior of individual kinesin molecules under two previously unexplored loading regimes: super-stall loads (>5 pN) and forward (plus-end directed) loads. Whereas some theories of kinesin function predict a reversal of directionality under high loads, we found that kinesin does not walk backwards under loads of up to 13 pN, probably because of an irreversible transition in the mechanical cycle. We also found that this cycle can be significantly accelerated by forward loads under a wide range of ATP concentrations. Finally, we noted an increase in kinesin’s rate of dissociation from the microtubule with increasing load, which is consistent with a load dependent partitioning between two recently described kinetic pathways: a coordinated-head pathway (which leads to stepping) and an independent-head pathway (which is static).

Background: Nivo is an inhibitory antibody against programmed death receptor-1 (PD-1), a regulator of antitumor immunity. Nivo is approved for treatment of unresectable or metastatic melanoma and disease progression (PD) following ipilimumab or, in BRAF V600 mutation–positive melanoma, following a BRAF inhibitor, and for metastatic squamous non-small cell lung cancer (NSCLC) following PD during or after platinum-based chemotherapy. Nivo and other immune checkpoint inhibitors are also being investigated in other tumor-types. nab-P is a novel taxane formulation and does not require prophylaxis with immunosuppressive steroids. It has demonstrated superior efficacy over control regimens in phase III studies of MBC, pancreatic cancer, and NSCLC. This open-label, 6-arm, multicenter phase I trial will evaluate the safety of Nivo with nab-P in 3 cancer types (2 arms/disease): MBC, advanced NSCLC (+ carboplatin), and advanced pancreatic cancer (± gemcitabine). The study design for the MBC portion is described below. Methods: Eligibility criteria include histologically/cytologically confirmed HER2-negative MBC; 1 prior chemotherapy for MBC, including an anthracycline unless clinically contraindicated; no relapse < 12 months after taxane adjuvant therapy; measurable disease by RECIST v1.1; ECOG performance status 0-1; adequate organ function; and preexisting peripheral neuropathy grade < 2. Patients (pts) with MBC will be treated in 2 arms: nab-P 100 mg/m2 on days 1, 8, and 15 of each 28-day cycle plus Nivo 3 mg/kg on days 1 and 15 starting at cycle 3 or nab-P 260 mg/m2 on day 1 of each 21-day cycle plus Nivo 5 mg/kg on day 15 starting at cycle 3. Pts will be treated until PD or allowed to continue treatment beyond RECIST v1.1–defined PD if they continue to meet study eligibility; do not have rapid PD or clinical deterioration or unacceptable toxicities; and can benefit from continuation of study treatment in the treating physician's opinion and will not delay an imminent intervention to prevent serious complications of PD. The primary endpoints of the study are the number of pts with dose-limiting toxicities (DLTs) in each treatment arm (part 1) and the percentage of pts with grade 3/4 treatment-emergent adverse events (TEAEs) or treatment discontinuation due to a TEAE (parts 1 and 2). Part 1 of the study will assess whether the starting dose of Nivo is deemed safe (≤ 1 DLT in 6 pts); otherwise, the Nivo dose will be de-escalated and assessed in a new cohort at the next lower dose level. The Nivo dose in combination with nab-P deemed safe in a treatment arm may be further assessed in part 2 of the study, with enrollment expanded to an additional ∼ 14 pts/arm (total of 20 Nivo-treated pts/arm). Secondary study endpoints include TEAEs leading to dose reduction, delay, interruption, or treatment discontinuation; progression-free survival; overall survival; disease control rate; overall response rate; and duration of response (per RECIST v1.1). Exploratory endpoints include tumor-associated PD-L1 expression, modulation of immune activation in the tumor and peripheral blood in response to Nivo treatment, Nivo serum levels, and development of anti-globulin antibodies. ClinicalTrials.gov identifier NCT02309177. Citation Format: Waterhouse D, Gutierrez M, Bekaii-Saab T, DeRosa W, Wainberg Z, George B, Duval Fraser C, Ko A, Pierce DW, Stergiopoulos S, Soliman H. nab-paclitaxel (nab-P) plus nivolumab (Nivo) in human epidermal growth factor receptor 2 (HER2)–negative recurrent metastatic breast cancer (MBC). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-01-07.

TPS475 Background: Nivo, an antibody against PD-1, is approved for unresectable or metastatic melanoma and metastatic squamous non-small cell lung cancer (NSCLC) after initial standard therapies. nab-P does not require immunosuppressive steroid premedication and is approved for treatment of metastatic breast cancer (MBC) after initial chemotherapy (CT) including an anthracycline, and for advanced NSCLC (+ carboplatin), and metastatic PC (+ Gem). This 6-arm, multicenter phase I trial will evaluate the safety of Nivo with nab-P— based therapy in MBC, advanced NSCLC, and advanced PC. The study design for the pancreatic portion (arm A: nab-P + Nivo; arm B: nab-P + Gem + Nivo) is described below. Methods: This 2-part study will identify dose-limiting toxicities (DLTs) in Part 1 and assess safety and explore antitumor activity in Parts 1 and 2 (Table). If Arm A, Part 1, is deemed safe (≤ 1 DLT in up to 6 pts), then Arm B, Part 1, will initiate enrollment. Regimens in Part 1 may be expanded in Part 2. Enrollment in Arm A, Part 1, is ongoing. ClinicalTrials.gov: NCT02309177. Clinical trial information: NCT02309177. [Table: see text]

The dielectric relaxation of a sperm whale ApoMb-DANCA complex is measured by the fluorescence dynamic Stokes shift method. Emission energy increases with decreasing temperature, suggesting that the relaxation activation energies of the rate-limiting motions either depend on the conformational substrate or different types of protein motions with different frequencies participate in the reaction. Experimental data suggest that there may be relaxations on a scale of

Inhibition of mitotic kinesins represents a novel approach for the discovery of a new generation of anti-mitotic cancer chemotherapeutics. We report here the discovery of the first potent and selective inhibitor of centromere-associated protein E (CENP-E) 3-chloro-N-{(1S)-2-[(N,N-dimethylglycyl)amino]-1-[(4-{8-[(1S)-1-hydroxyethyl]imidazo[1,2-a]pyridin-2-yl}phenyl)methyl]ethyl}-4-[(1-methylethyl)oxy]benzamide (GSK923295; 1), starting from a high-throughput screening hit, 3-chloro-4-isopropoxybenzoic acid 2. Compound 1 has demonstrated broad antitumor activity in vivo and is currently in human clinical trials.

Kinesin is a two-headed motor protein that powers organelle transport along microtubules. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.

Background and Aim: Advanced pancreatic cancer is both deadly and difficult to treat with success. Here, we disseminate the results of comprehensive preclinical studies which laid a strong foundation for the rapid bench to bedside development of nab-paclitaxel, an albumin-bound formulation of paclitaxel, in combination with gemcitabine, a regimen recently approved by U.S. Food and Drug Administration as a first-line treatment for patients with metastatic pancreatic cancer. Materials and Methods: We enrolled a total of 650 mice with established pancreatic tumors originated from a collection of patient-derived pancreatic cancer xenografts. The present study investigated the anti-tumor activity, survival advantage and mechanism of action of nab-paclitaxel or Cremophor EL™ (CreEL)-based paclitaxel monotherapy and in combination with gemcitabine. Results: When tested in mice with subcutaneous tumors originating from 11 separate individual patient xenografts, nab-paclitaxel plus gemcitabine treatment demonstrated superior tumor regression response, robustly depleted the tumor desmoplatic stroma, leading to enhanced gemcitabine uptake (2.8-fold) in the tumor compared to tumors in the gemcitabine alone treated mice. In orthotopic models, nab-paclitaxel treatment leads to an average of 3.64-fold decrease in primary tumor volumes compared to CreEL-based paclitaxel. Intra-tumor stromal collapse combined with decreased tumor cell proliferation was clearly evident in the primary tumors of nab-paclitaxel treated mice compared to CreEL-based paclitaxel, when the mice were sacrificed immediately after five consecutive day's treatment or three weeks after the final dose of the agents. In a highly aggressive orthotopic model, nab-paclitaxel plus gemcitabine treatment prevented primary tumor progression, and metastatic spread to liver, lymph nodes and diaphragm. While CreEL-based paclitaxel plus gemcitabine treatment failed to enhance mouse survival compared to gemcitabine monotherapy, the nab-paclitaxel plus gemcitabine combination proved statistically significant (p=0.0133) in enhancing survival. nab-paclitaxel monotherapy demonstrated statistically significant survival advantage compared to CreEL-based paclitaxel monotherapy (p=0.0072). Remarkably, nab-paclitaxel monotherapy was equivalent to nab-paclitaxel plus gemcitabine in providing survival advantage in a highly aggressive metastatic model of pancreatic cancer. Conclusion: Our results demonstrated that co-treatment with nab-paclitaxel and gemcitabine resulted in superior tumor regression response, stromal depletion and enhanced intra-tumoral gemcitabine uptake compared with either single agent alone. nab-paclitaxel demonstrated superior anti-tumor activity and provided a statistically significant survival advantage compared to CreEL-based paclitaxel. Our results provide further rationale for future preclinical and clinical trials in pancreatic cancer using nab-paclitaxel as a backbone therapy in combination with novel experimental and targeted agents. Acknowledgements: The study was supported by funding from Celgene Corporation and AACR-Stand Up To Cancer Dream Team Translational Cancer Research Grant (SU2C-AACR-DT0509). Citation Format: N.V Rajeshkumar, Shinichi Yabuuchi, Shweta G. Pai, Scott Bateman, Ellen Filvaroff, Daniel W. Pierce, Carla Heise, Daniel D. Von Hoff, Anirban Maitra, Manuel Hidalgo. Highlights of innovative preclinical studies which guided the rapid bench to bedside development of nab-paclitaxel plus gemcitabine combination for the treatment of pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-94. doi:10.1158/1538-7445.AM2014-LB-94

The chemotaxis signal protein CheY of enteric bacteria shuttles between transmembrane methyl-accepting chemotaxis protein (MCP) receptor complexes and flagellar basal bodies [1]. The basal body C-rings, composed of the FliM, FliG and FliN proteins, form the rotor of the flagellar motor [2]. Phosphorylated CheY binds to isolated FliM [3] and may also interact with FliG [4], but its binding to basal bodies has not been measured. Using the chemorepellent acetate to phosphorylate and acetylate CheY [5], we have measured the covalent-modification-dependent binding of a green fluorescent protein–CheY fusion (GFP–CheY) to motor assemblies in bacteria lacking MCP complexes by evanescent wave microscopy [6]. At acetate concentrations that cause solely clockwise rotation, GFP–CheY molecules bound to native basal bodies or to overproduced rotor complexes with a stoichiometry comparable to the number of C-ring subunits. GFP–CheY did not bind to rotors lacking FliM/FliN, showing that these subunits are essential for the association. This assay provides a new means of monitoring protein–protein interactions in signal transduction pathways in living cells.

Conventional kinesin is capable of long-range, processive movement along microtubules, a property that has been assumed to be important for its role in membrane transport. Here we have investigated whether the Caenorhabditis elegans monomeric kinesin unc104 and the sea urchin heteromeric kinesin KRP85/95, two other members of the kinesin superfamily that function in membrane transport, are also processive. Both motors were fused to green fluorescent protein, and the fusion proteins were tested for processive ability using a single-molecule fluorescence imaging microscope. Neither unc104-GFP nor KRP85/95-GFP exhibited processive movement (detection limit approximately 40 nm), although both motors were functional in multiple motor microtubule gliding assays (v = 1760 +/- 540 and 202 +/- 37 nm/s, respectively). Moreover, the ATP turnover rates (5.5 and 3.1 ATPs per motor domain per second, respectively) are too low to give rise to the observed microtubule gliding velocities, if only a single motor were driving transport with an 8 nm step per ATPase cycle. Instead, the results suggest that these motors have low duty cycles and that high processivity may not be required for efficient vesicle transport. Conventional kinesin's unusual processivity may be required for efficient transport of protein complexes that cannot carry multiple motors.

The cellular slime mold Dictyostelium discoideum is an attractive system for studying the roles of microtubule-based motility in cell development and differentiation. In this work, we report the first molecular characterization of kinesin-related proteins (KRPs) in Dictyostelium. A PCR-based strategy was used to isolate DNA fragments encoding six KRPs, several of which are induced during the developmental program that is initiated by starvation. The complete sequence of one such developmentally regulated KRP (designated K7) was determined and found to be a novel member of the kinesin superfamily. The motor domain of K7 is most similar to that of conventional kinesin, but unlike conventional kinesin, K7 is not predicted to have an extensive α-helical coiled-coil domain. The nonmotor domain is unusual and is rich in Asn, Gln, and Thr residues; similar sequences are found in other developmentally regulated genes inDictyostelium. K7, expressed in Escherichia coli, supports plus end–directed microtubule motility in vitro at a speed of 0.14 μm/s, indicating that it is a bona fide motor protein. The K7 motor is found only in developing cells and reaches a peak level of expression between 12 and 16 h after starvation. By immunofluorescence microscopy, K7 localizes to a membranous perinuclear structure. To examine K7 function, we prepared a null cell line but found that these cells show no gross developmental abnormalities. However, when cultivated in the presence of wild-type cells, the K7-null cells are mostly absent from the prestalk zone of the slug. This result suggests that in a population composed largely of wild-type cells, the absence of the K7 motor protein interferes either with the ability of the cells to localize to the prestalk zone or to differentiate into prestalk cells.

Kinesin is a dimeric motor protein that can move along a microtubule for several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in processive movement. Recent models have suggested that the neck enables head-to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed processivity using a fluorescence microscope that can visualize single kinesin–GFP molecules moving along a microtubule. Our results show that replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduce the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement. Furthermore, we show that deleting the neck coiled coil decreases processivity 10-fold, but surprisingly does not abolish it. We also demonstrate that processivity is increased by threefold when the neck helix is elongated by seven residues. These results indicate that structural features of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.

Publisher Summary This chapter discusses the features of fluorescence microscopes for single molecule detection and describes the design and construction of the low background total internal reflection fluorescence microscope that are used in experiments. In addition, the unusual properties of green fluorescent protein (GFP) compared to small organic fluorophores have been discussed, with particular attention to its behavior under single molecule imaging conditions. The chapter also summarizes the applications of single-molecule GFP imaging. It utilizes single-molecule detection of kinesin-GFP, expressed by conventional means or by in vitro translation, to develop an assay for visualizing single-molecule motility of this motor protein. GFP fusions of many molecules of interest are already available, and more appear on a daily basis. Thus, it should be possible to extend these studies to other moving-enzyme systems such as helicases, polymerases, and ribosomes, and more generally to the quantitation of affinities and rate parameters of protein–protein interactions. Preliminary evidence suggests that, in some circumstances, single-molecule imaging of GFP fusion proteins in living cells may be possible.