Combination Therapy of Selinexor with Bortezomib or Carfilzomib Overcomes Drug Resistance to Proteasome Inhibitors (PI) in Human Multiple Myeloma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3048-3048
Author(s):  
Daniel M Sullivan ◽  
Trinayan Kashyap ◽  
Jana L Dawson ◽  
Yosef Landesman ◽  
Steven Grant ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Protein Expression ◽  
Light Chain ◽  
Immunofluorescence Microscopy ◽  
Single Agent ◽  
Vehicle Control ◽  
Heavy Isotope ◽  
Employment Equity ◽  
Equity Ownership

Abstract Purpose: Drug resistance is the greatest obstacle to the successful treatment of multiple myeloma (MM). We investigated whether the clinical XPO1 inhibitor selinexor (KPT-330), when combined with bortezomib or carfilzomib, could overcome proteasome inhibitor (PI) resistance in myeloma. Experimental Design: PI-resistant human MM cell lines 8226-B25 and U266-PSR were treated with the XPO1 inhibitors selinexor or KOS-2464 in combination with bortezomib or carfilzomib and assayed for apoptosis and viability. Mice challenged with PI-resistant human MM cells (U266-PSR) were treated with selinexor +/- bortezomib. CD138+/light-chain+ MM cells from PI-refractory MM patients were treated with selinexor +/- bortezomib or selinexor +/- carfilzomib and assayed for apoptosis. All experiments were compared to the standard of care, bortezomib therapy. IkBα-protein was assayed by Western blot and immunofluorescence microscopy and IkBα-NFkB-complex formation by proximity ligation assay. IkBα protein knockdown in human MM cells by siRNA was performed to determine the mechanism of selinexor inhibitor action. Further analysis of selinexor/bortezomib treatment on intra-cellular protein levels and intra-cellular localization was performed by lysine and N-terminal labeling with six-plex tandem mass tags (heavy isotope) and assayed by LC-MS/MS discovery proteomics. Results: Selinexor in combination with bortezomib or carfilzomib decreased viability and induced apoptosis in PI-resistant MM cells. Resistant MM cell lines were up to 10-fold resistant to single agent bortezomib or carfilzomib when compared to parental cells. The combination of the XPO1 inhibitors selinexor or KOS-2464 sensitized drug resistant cells to bortezomib (P < 0.02) and carfilzomib (P < 0.005) when compared to single agents. Selinexor and bortezomib inhibited PI-resistant MM tumor growth and increased survival with minimal toxicity in NOD/SCID-g mice. Bone marrow mononuclear cells isolated and treated with selinexor or KOS-2464 and bortezomib or carfilzomib from newly diagnosed (n=8), relapsed (n=5), and bortezomib (n=8) and carfilzomib (n=6) refractory MM patient samples were all sensitized by selinexor and KOS-2464 to bortezomib (P < 0.043) and carfilzomib (P < 0.044) as shown by increased apoptosis. Normal, non-myeloma CD138/light-chain double-negative patient cells were not sensitized to apoptosis by XPO1 inhibitors. Immunofluorescence microscopy of IkBα in 8226-B25 PI-resistant cells showed an increase in IkBα after treatment with selinexor/bortezomib as compared with vehicle control or single agent bortezomib or selinexor. Nuclear IκBα was also increased by selinexor treatment. IkBα protein expression was increased by bortezomib (70%) and selinexor (50%) versus control. The selinexor/bortezomib combination increased IkBα protein (212%) as compared to vehicle control or single agent bortezomib or selinexor. Similar results were found in drug-naïve 8226 and U226 cells, as well as PI-resistant 8226-B25 and U225-PSR cells. The increase in nuclear IkBα after selinexor treatment was confirmed by ImageStream flow cytometry. Selinexor/bortezomib therapy significantly increased IkBα-NFkB-complexes in PI-resistant MM cells. Selinexor in combination with bortezomib increased proximity co-localization of NFkB and IkBα without affecting XPO1 protein expression after 4 hours of drug treatment. Analysis of the number of NFkB-IkBα foci/binding showed that selinexor/bortezomib increased the number of foci in the nucleus versus untreated control or single agent selinexor or bortezomib (P ≤ 0.00077). IkBα knockdown reduced selinexor-induced cytotoxicity in both IM-9 (9.5-fold) and 8226 (12.3 to 25.4-fold) human MM cells. Intracellular protein analysis by heavy isotope labeling and LC-MS/MS showed changes in several signaling pathways including p53, MAPK, VEGF and angiopoietin, IL-1, HMGB1/TLR and APRIL and BAFF as well as those related to NFkB signaling. Conclusion: Selinexor, when used in combination with bortezomib or carfilzomib has the potential to overcome PI drug resistance in MM. Disclosures Kashyap: Pharma: Employment. Landesman:Karyopharm Therapeutics: Employment. Kauffman:Karyopharm: Employment, Equity Ownership. Shacham:Karyopharm: Employment, Equity Ownership.

Impact of Baseline Free Light Chain Ratio (rFLC) On Clinical Outcomes and Change in rFLC During Treatment in Patients with Relapsed/Refractory Multiple Myeloma Treated with Pegylated Liposomal Doxorubicin Plus Bortezomib or Bortezomib Alone.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4778-4778
Author(s):  
Valeria Magarotto ◽  
Anil Londhe ◽  
Keith C. Lantz ◽  
Colin Lowery ◽  
Pieter Sonneveld ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Outcomes ◽  
Light Chain ◽  
Liposomal Doxorubicin ◽  
Pegylated Liposomal Doxorubicin ◽  
Free Light Chain ◽  
Controlled Study ◽  
Employment Equity ◽  
Ortho Biotech ◽  
Equity Ownership

Abstract Abstract 4778 Background The serum immunoglobulin-free light chain (sFLC) assay measures levels of free κ and » immunoglobulin light chains. In multiple myeloma (MM), similar to other plasma cell dyscrasias, a secretory dysfunction causes an abnormal sFLC (κ/») ratio (rFLC). The rFLC (normal range: 0.26-1.65) is a requirement for documenting stringent complete remission (sCR) and can be a prognostic indicator in newly diagnosed multiple myeloma (Dispenzieri et al. for the International Myeloma Working Group, Leukemia, 2009). As part of a randomized controlled study in MM (Orlowski et al., JCO 2007), we previously have shown that the normalization of rFLC after the first 2 cycles of pegylated liposomal doxorubicin (PLD) + bortezomib (B) or B alone is associated with a prolonged time to progression (TTP) and higher response rate (RR) among patients with relapsed/refractory MM (Orlowski et al., Blood 2007, abstract #2735). In this analysis, we examined how baseline ratios of sFLC impacted clinical outcomes (TTP and RR), and if sFLC ratios changed over the course of treatment in patients receiving PLD + B or B alone. Methods Patients with ≥1 prior therapy were randomized to receive PLD at 30 mg/m2 on day 4 with B at 1.3 mg/m2 on Days 1, 4, 8, and 11, or B alone on this same dose and schedule, for up to eight 21-day cycles, or at least 2 cycles beyond complete response (CR) or optimal response, unless disease progression or unacceptable toxicities occurred. Serial serum κ/» measurements were made prior to the start of therapy and at the end of each cycle throughout the entire study using an immunoassay (Freelite, The Binding Sites, Birmingham, UK). Results Sera from 491 patients were available for these analyses: 31 patients (6.3%) had a normal baseline rFLC, while 460 patients (93.6%) had an abnormal baseline rFLC (<0.26 or >1.65). Among patients with a normal baseline rFLC, 14 received B and 17 received PLD + B. The RR (≥partial remission) was 5/14 (35.7%) and 5/17 (29.4%), respectively (P=0.73). Mean TTP was 297 days for B vs. not reached for PLD + B (P=0.93, HR 0.92, CI 95%). In patients with an abnormal baseline rFLC, 232 received B and 228 received PLD + B. The RR was 95/232 (40.9%) and 107/228 (46.9%), respectively (P=0.19). TTP was longer and statistically significant for patients who received PLD + B (282 days) vs. B alone (180 days; P=0.001, HR 1.76, CI 95%). For the overall population, TTP was 297 vs. 218 days (P=0.11, HR 0.51, CI 95%) and RR was 10/31 (32.3%) vs 202/460 (43.9%) for normal vs abnormal baseline rFLC groups, respectively. During the course of therapy, there was a trend for an increase in the percentage of patients in whom the baseline rFLC changed from abnormal to normal (Table). This trend occurred in earlier treatment cycles and reached a plateau in later cycles. When only patients who had completed at least 4 cycles of therapy were examined, the same trend was observed (not shown). Conclusion This study showed a correlation between the baseline rFLC and outcomes (TTP and RR) in patients with relapsed/refractory MM treated with PLD + B or B alone. Similar to the overall study results, combination therapy demonstrated better outcomes over B alone in patients with abnormal rFLC at baseline. Regardless of treatment, patients with normal rFLC at baseline had longer TTP but lower RR than patients with abnormal rFLC at baseline, which was possibly due to the small number of patients with normal rFLC at baseline. Disclosures: Londhe: Centocor Ortho Biotech Services, LLC: Employment, Equity Ownership. Lantz:Centocor Ortho Biotech Services, LLC: Employment. Lowery:Centocor Ortho Biotech Services, LLC: Employment, Equity Ownership. Sonneveld:Johnson & Johnson: Consultancy. Bladé:Johnson & Johnson / Janssen-Cilag: Honoraria; Schering-Plough: Honoraria.

The Epitope Targeted by Apoptosis-Inducing ICAM-1 Antibody B11 Is Highly Expressed in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4897-4897
Author(s):  
Markus Hansson ◽  
Niina Veitonmäki ◽  
Anders Lindblom ◽  
Björn Frendéus
Keyword(s):  
Multiple Myeloma ◽  
Drug Resistance ◽  
Cell Adhesion ◽  
Disease Progression ◽  
Plasma Cells ◽  
Human Tumor ◽  
University Hospital ◽  
Employment Equity ◽  
New Drug ◽  
Equity Ownership

Abstract Abstract 4897 Complex adhesive and non-cognate interactions participate in multiple myeloma disease progression, resistance to apoptosis, and development of drug resistance. In spite of significant recent attempts to develop new drug classes targeting both myeloma and its microenvironment, multiple myeloma remains an incurable disease warranting development of more effective therapies. Applying novel combined target and drug discovery methodology we isolated a human tumor cell apoptosis-inducing antibody (IgG B11) targeting ICAM-1, as previously described. ICAM-1 is a cell adhesion molecule strongly implicated in myeloma pathophysiology, both regarding bone marrow stromal cell mediated disease progression and cell adhesion mediated drug resistance. We here characterize B11 epitope expression by multicolor FACS analysis in 25 patients investigated for multiple myeloma referred to Department of Hematology, Lund University Hospital, Lund, Sweden and 5 healthy controls. The B11 epitope was highly expressed in plasma cells in 5 of 5 patients with myeloma and in 1 of 1 patient with AL (light chain) amyloidosis. A comprehensive preclinical program assessing IgG B11 anti-myeloma activity and evaluating IgG B11 safety has been conducted. Based on these studies, and having received an investigational new drug (IND) approval from the U.S. Food and Drug Administration (FDA), clinical phase I trials with IgG B11 will soon commence. Disclosures Veitonmäki: BioInvent International: Employment. Lindblom:BioInvent International: Employment, Equity Ownership. Frendéus:BioInvent International AB: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

A Phase I/II Trial of the KSP Inhibitor ARRY-520 In Relapsed/Refractory Multiple Myeloma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1959-1959 ◽  
Author(s):  
Jatin J Shah ◽  
Jeffrey A. Zonder ◽  
Adam Cohen ◽  
Donna Weber ◽  
Sheeba Thomas ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Dose Escalation ◽  
Research Funding ◽  
Phase 1 ◽  
Single Agent ◽  
Clinical Activity ◽  
Employment Equity ◽  
Heavily Pretreated ◽  
Equity Ownership ◽  
Ksp Inhibitor

Abstract Abstract 1959 Background: Kinesin Spindle Protein (KSP) is required for cell cycle progression through mitosis. Inhibition of KSP induces mitotic arrest and cell death. ARRY-520 is a potent, selective KSP inhibitor. Cancers such as multiple myeloma (MM) which depend on the short-lived survival protein MCL-1 are highly sensitive to treatment with ARRY-520. ARRY-520 shows potent activity in preclinical MM models, providing a strong rationale for its clinical investigation in this disease. Methods: This Phase 1 study was designed to evaluate the safety and pharmacokinetics (PK) of ARRY-520 administered intravenously (IV) on Day 1 and Day 2 q 2 weeks without/with granulocyte-colony stimulating factor (G-CSF). Patients (pts) with relapsed/refractory (RR) MM with 2 prior lines of therapy (including both bortezomib and an immunomodulatory agent, unless ineligible for or refusing to receive this therapy) were eligible. Cohorts of at least 3 pts were enrolled in a classical 3 + 3 dose escalation design. Pts were treated for 2 cycles (4 weeks) to evaluate safety prior to dose escalation. Results: Twenty five pts have been treated to date, with a median age of 60 years (range 44–79) and a median of 5 prior regimens (range 2–16). All pts received prior bortezomib or carfilzomib, 21 pts received prior lenalidomide, 17 pts prior thalidomide, and 18 pts had a prior stem cell transplant. Pts received ARRY-520 without G-CSF at 1 mg/m2/day (n = 3), and at 1.25 mg/m2/day (n = 7, 6 evaluable). A dose-limiting toxicity (DLT) of Grade 4 neutropenia was observed at 1.25 mg/m2/day, and this was considered the maximum tolerated dose (MTD) without G-CSF. As neutropenia was the DLT, dose escalation with prophylactic G-CSF support was initiated, at doses of 1.5 mg/m2/day (n = 7, 6 evaluable), 2.0 mg/m2/day (n = 6) and 2.25 mg/m2/day (n = 2) with G-CSF. Both the 2.0 mg/m2/day and 2.25 mg/m2/day dose levels were determined to be non-tolerated, with DLTs of febrile neutropenia (FN) (2 pts at 2.0 mg/m2/day and both pts at 2.25 mg/m2/day) and Grade 3 mucositis (both pts at 2.25 mg/m2/day). One out of 6 evaluable pts at 1.5 mg/m2/day also developed a DLT of FN. In an attempt to optimize the Phase 2 dose, an intermediate dose level of 1.75 mg/m2/day with G-CSF is currently being evaluated. The most commonly reported treatment-related adverse events (AEs) include those observed with other KSP inhibitors, such as hematological AEs (thrombocytopenia, neutropenia, anemia, leukopenia), fatigue, mucositis and other gastro-intestinal AEs. Pts displayed linear PK, a low clearance and a moderate volume of distribution, with moderate-to-high inter-individual variability in PK parameters. The median terminal elimination half life is 65 hours. The preliminary efficacy signal as a single agent is encouraging with 2 partial responses (PR) observed to date per IMWG and EBMT criteria in a heavily pretreated population (23 evaluable pts). A bortezomib-refractory pt with 8 prior lines of therapy, including a tandem transplant, treated at 1 mg/m2/day of ARRY-520 obtained a PR after Cycle 6, with urine protein and kappa light chain levels continuing to decline over time. He remains on-study after 15 months of ARRY-520 treatment. A pt with 2 prior lines of therapy, including prior carfilzomib, has obtained a PR after Cycle 8 at 2 mg/m2/day of ARRY-520, and she is currently ongoing after 4.5 months on therapy. Fifteen pts had a best response of stable disease (SD), including 1 pt with a thus far unconfirmed minimal response, and 6 had progressive disease. A total of 10 pts (43%) achieved a PR or SD lasting > 12 weeks. Several additional pts have shown other evidence of clinical activity, with decrease in paraproteins, increase in hemoglobin levels and regression of plasmacytomas. The median number of cycles is 4 (range 1–28+). Treatment activity has not correlated with any baseline characteristics or disease parameters to date. Conclusions: : The selective KSP inhibitor ARRY-520 has been well tolerated, and shows promising signs of single agent clinical activity in heavily pretreated pts with RR MM. Prophylactic G-CSF has enabled higher doses to be tolerated. No cardiovascular or liver enzyme toxicity has been reported. Enrollment is ongoing at 1.75 mg/m2/day with G-CSF support, and a planned Phase 2 part of the study will be initiated as soon as the MTD is determined. Complete Phase 1 data will be disclosed at the time of the meeting. Disclosures: Shah: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Millennium: Research Funding. Off Label Use: Revlimid (lenalidomide) in combination with dexamethasone is indicated for the treatment of multiple myeloma patients who have received at least one prior therapy. Zonder:Millennium: Consultancy, Myeloma and Amyloidosis Patient Day Symposium – Corporate support from multiple sponsors for a one-day educational event, Research Funding; Celgene:; Novartis:; Proteolix: . Weber:novartis-unpaid consultant: Consultancy; Merck- unpaid consultant: Consultancy; celgene- none for at least 2 years: Honoraria; millenium-none for 2 years: Honoraria; celgene, Millenium, Merck: Research Funding. Wang:Celgene: Research Funding; Onyx: Research Funding; Millenium: Research Funding; Novartis: Research Funding. Kaufman:Celgene: Consultancy, Honoraria, Research Funding; Millenium: Consultancy, Honoraria; Merck: Research Funding; Genzyme: Consultancy. Walker:Array Biopharma: Employment, Equity Ownership. Freeman:Array Biopharma: Employment, Equity Ownership. Rush:Array Biopharma: Employment, Equity Ownership. Ptaszynski:Array Biopharma: Consultancy. Lonial:Millennium, Celgene, Bristol-Myers Squibb, Novartis, Onyx: Advisory Board, Consultancy; Millennium, Celgene, Novartis, Onyx, Bristol-Myers Squibb: Research Funding.

A Selective PIM Kinase Inhibitor Is Highly Active In Multiple Myeloma: Mechanism of Action and Signal Transduction Studies

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4084-4084 ◽  
Author(s):  
Veerendra Munugalavadla ◽  
Leanne Berry ◽  
Yung-Hsiang Chen ◽  
Gauri Deshmukh ◽  
Jake Drummond ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
Combination Treatment ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Myeloma Cell ◽  
Negative Regulator ◽  
Combination Drug ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 4084 Related work from our group has shown the therapeutic utility of PIM inhibition in multiple myeloma cell lines, xenografts, and primary patient samples (Ebens A. et al., ASH 2010 submitted abstr.). In this study we provide detailed mechanistic findings to show that PIM kinase inhibition co-regulates several important elements of the PI3K/AKT/mTOR pathway, resulting in significant synergy for combination drug treatments. The PIM kinases are a family of 3 ser/thr growth factor- & cytokine-induced proteins hypothesized to have redundant survival and growth functions. GNE-652 is a pan-PIM kinase inhibitor with picomolar biochemical potencies and an excellent kinase selectivity profile. Myeloma cell lines exhibit sensitivity to single agent PIM inhibition and a striking synergy in combination with the PI3K inhibitor GDC-0941. Cells respond to this combination with cell cycle arrest and marked apoptosis in vitro. We tested a panel of selective PI3K/AKT/mTOR inhibitors and found PI3K and AKT inhibitors showed the greatest extent of synergy with GNE-652, whereas mTOR inhibitors were synergistic to a lesser extent. These results suggest that PIM signaling converges on both TORC1 and AKT to generate these differential synergies. BAD is a negative regulator of both Bcl-2 and Bcl-XL, and we were able to confirm previous reports that AKT and PIM cooperate to inactivate BAD (Datt et al., 1997; Yan et al., 2003). Pim has been shown to potentially inactivate PRAS40, a negative regulator of TORC1 (Zhang et al., 2009). We demonstrate that PIM or PI3K inhibition caused a loss of phosphorylation on PRAS40 and results in a physical association of PRAS40 and TORC1 and a decrease in phosphorylated p70S6K and S6RP. These reductions were apparent in 7 of 7 cell lines assayed and enhanced by the combination of PI3K and PIM inhibition in these cell lines. Consistent with prior reports (Hammerman et al., 2005), we show that a second node of convergence between PIM and TORC1 is 4E-BP1. Both GDC-0941 and GNE-652 treatments reduced phosphorylation of 4E-BP1 in 7 of 7 myeloma cell lines. Since dephosphorylated 4E-BP1 competes with eIF4G for the mRNA cap binding factor eIF4E, we assayed immunoprecipitates of eIF4E for the presence of eIF4G and 4E-BP1 and observed increased BP1 and decreased 4G. The combination treatment significantly enhanced the loss of 4G relative to either single agent, and importantly, even at 5× the IC50 concentrations for single agents, combination drug treatment achieved greater extent of effect than single agent treatment. Thus PI3K and PIM pathways are redundant at the level of cap-dependent translational initiation mediated by eIF4E. It has been hypothesized a subset of mRNAs are particularly sensitive to inhibition of cap-dependent translation, and that this includes a number of oncogenes such as cyclin D1. We assayed global protein synthesis in MM1.s cells using 35S-methionine and as expected we observed only a modest ≂∼f20% decrease caused by either GNE-652 or GDC-0941 and this decrease was not enhanced by combination treatment. However, we noted across 7 different myeloma cell lines, strong decreases in levels of cyclin D1 that were enhanced by combination treatment. In summary, we have identified several points at which PIM and PI3K/AKT/mTOR converge to provide synergistic apoptosis in multiple myeloma cell lines. These results provide the rationale for further preclinical development of PIM inhibitors and provide the basis for a possible clinical development plan in multiple myeloma. Disclosures: Munugalavadla: Genentech: Employment, Equity Ownership. Berry:Genentech: Employment, Equity Ownership. Chen:Genentech: Employment, Equity Ownership. Deshmukh:Genentech: Employment, Equity Ownership. Drummond:Genentech: Employment, Equity Ownership. Du:Genentech: Employment, Equity Ownership. Eby:Genentech: Employment, Equity Ownership. Fitzgerald:Genentech: Employment, Equity Ownership. S.Friedman:Genentech: Employment, Equity Ownership. E.Gould:Genentech: Employment, Equity Ownership. Kenny:Genentech: Employment, Equity Ownership. Maecker:Genentech: Employment, Equity Ownership. Moffat:Genentech: Employment, Equity Ownership. Moskalenko:Genentech: Employment, Equity Ownership. Pacheco:Genentech: Employment, Equity Ownership. Saadat:Genentech: Employment, Equity Ownership. Slaga:Genentech: Employment, Equity Ownership. Sun:Genentech: Employment, Equity Ownership. Wang:Genentech: Employment, Equity Ownership. Yang:Genentech: Employment, Equity Ownership. Ebens:Genentech Inc: Employment, Equity Ownership.

Detection and Quantification of Cereblon Protein and mRNA in Multiple Myeloma Cell Lines and Primary CD138+multiple Myeloma Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4043-4043
Author(s):  
Anita K Gandhi ◽  
Herve Avet-Loiseau ◽  
Michelle Waldman ◽  
Anjan Thakurta ◽  
Sharon L Aukerman ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Western Blot ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Full Length ◽  
Employment Equity ◽  
Equity Ownership ◽  
Protein Variants

Abstract Abstract 4043 Background: Cereblon (CRBN), a component of the DDB1-CUL4A-Roc1 ubiquitin ligase complex, has been identified as a target of the immunomodulatory agents thalidomide, lenalidomide, and pomalidomide (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011; Ito et al. Science. 2010.). CRBN binding by these agents mediates their anti-proliferative effects in multiple myeloma (MM) cells (Lopez-Girona et al. Leukemia. 2012; Zhu et al. Blood. 2011). However, the role of CRBN quantification as a marker for disease responsiveness or resistance to these drugs remains to be fully defined. Furthermore, it is unclear whether measuring mRNA or protein expression is the best approach for development of a quantitative CRBN expression assay. In order to define the optimal assay approach, we have studied CRBN mRNA and protein expression in MM cell lines (n=20) and MM patient samples. Methods: CRBN isoform mapping was undertaken using a nested PCR approach and Sanger sequencing. Commercially available and newly generated rabbit anti-CRBN antibodies were characterized with recombinant human CRBN protein and MM cell line extracts via western blot analysis. Results: Our data show that in addition to the transcript for full length protein (GenBank Accession NM_016302.3), in MM cells there are at least 6 alternatively spliced isoforms of CRBN as depicted in Figure 1. Five of the 6 CRBN isoforms (CRBN-003, -004, -005, -006, and -007) contain novel splice junctions not previously described. In addition, 3 of the identified transcripts (CRBN-002, -003, and -005) contain in-frame ORFs, suggesting they encode variants of CRBN protein. Of note, exon 10, which contains a portion of the IMiD-binding domain, is not present in CRBN-002. The functional consequence of CRBN-002 remains to be elucidated, but may be a marker of drug resistance. In order to measure CRBN protein levels, we developed and characterized three rabbit monoclonal antibodies to CRBN including antibody CRBN65, which has the potential to discriminate between the different CRBN protein products, including CRBN-002 by western blot analysis. Additionally, we compared 8 commercially available CRBN antibodies. Western blot analysis of cell lines with commercial and newly developed antibodies identified full length protein at 51 kD. Most commercial antibodies also identified multiple bands of other sizes which may represent CRBN protein variants; however, many are likely non-specific bands as they are larger than full-length CRBN. Conclusion: We have identified novel splice variants of CRBN from MM cell lines and primary tumor samples. The structure of the isoforms and their potential ability to be translated into several protein variants of CRBN reflect the complex regulation of the CRBN gene. These data suggest that accurate quantification of CRBN mRNA level in clinical studies may require measurement of both full-length CRBN mRNA as well as other mRNA isoforms. Currently available primers and gene expression arrays are not capable of identifying and/or resolving the complex set of CRBN isoforms present in cells. These data also demonstrate that CRBN65 is a highly specific and sensitive antibody that could be used for detection of CRBN and its key variants. Taken together, our data emphasize the importance for developing standardized reagents and assays for both mRNA and protein level measurement of CRBN before using them as markers for clinical response or resistance. Disclosures: Gandhi: Celgene Corp: Employment, Equity Ownership. Waldman:Celgene Corp: Employment, Equity Ownership. Thakurta:Celgene Corp: Employment, Equity Ownership. Aukerman:Celgene Corp: Employment, Equity Ownership. Chen:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Rychak:Celgene Corp: Employment, Equity Ownership. Miller:Celgene Corp: Employment, Equity Ownership. Gaidarova:Celgene Corp: Employment, Equity Ownership. Gonzales:Celgene Corp: Employment, Equity Ownership. Cathers:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Daniel:Celgene Corporation: Employment. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

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

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4255-4255 ◽  
Author(s):  
Ka Tat Siu ◽  
Homare Eda ◽  
Loredana Santo ◽  
Janani Ramachandran ◽  
Miroslav Koulnis ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Protein Expression ◽  
Cell Lines ◽  
Positive Feedback ◽  
Research Funding ◽  
Cytotoxic Effect ◽  
Feedback Mechanism ◽  
Employment Equity ◽  
Equity Ownership ◽  
Rpmi 8226

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

Results from Phase 1/2 Trial of Tagraxofusp in Combination with Pomalidomide and Dexamethasone in Relapsed or Refractory Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3145-3145 ◽  
Author(s):  
Paul G. Richardson ◽  
Myo Htut ◽  
Cristina Gasparetto ◽  
Jeffrey A. Zonder ◽  
Thomas G. Martin ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Research Funding ◽  
Phase 1 ◽  
Single Agent ◽  
Advisory Board ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Background: The bone marrow microenvironment of many multiple myeloma (MM) patients contains high levels of CD123-expressing plasmacytoid dendritic cells (pDCs). These pDCs have been shown to augment MM growth and contribute to drug resistance (Chauhan, et al., Cancer Cell, 2009). Tagraxofusp, a novel CD123 targeted therapy, has demonstrated high levels of anti-tumor activity in patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive CD123+ malignancy of pDC origin. Tagraxofusp demonstrated potent in vitro and in vivo activity against MM cell lines and primary tumor samples via both a direct anti-MM effect and indirect pDC-targeting effect (Ray, et al., Leukemia, 2017), as well as demonstrating synergy in these systems when used in combination with traditional MM therapies including pomalidomide (POM). As such, targeting pDCs with tagraxofusp may offer a novel therapeutic approach in MM. Methods: This multicenter, single arm Phase 1/2 trial enrolled patients with relapsed or refractory (r/r) MM and tested two different doses of tagraxofusp (7 or 9 mcg/kg). Patients received tagraxofusp as a daily IV infusion for days 1-5 of a 28-day cycle as a single agent for the initial run-in cycle (cycle 0) and in combination with standard doses/administration of POM and dexamethasone (DEX) in cycles 1 and beyond. Objectives included evaluation of safety and tolerability, identification of the maximum tolerated or tested dose, and efficacy. Results: 9 patients with r/r MM received tagraxofusp (7 mcg/kg, n=7; 9 mcg/kg, n=2). 5 males, median age 65 years (range: 57-70), median 3 prior therapies (range 2-6). Median follow-up was 12 months (range: 7 - 19). The most common treatment-emergent AEs (TEAEs) were hypoalbuminemia 67% (6/9); chills, fatigue, insomnia, nausea and pyrexia each 56% (5/9); and dizziness, headache, hypophosphatemia, and thrombocytopenia each 44% (4/9). The most common grade 3 and 4 TEAEs were thrombocytopenia 44% (4/9) and neutropenia 33% (3/9). No grade 5 events reported. 5 patients treated with tagraxofusp and POM+DEX had a partial response (PR) after tumor evaluation. These patients demonstrated a rapid decrease in a set of myeloma-related laboratory values from pre-tagraxofusp treatment levels after the first combination cycle of tagraxofusp and POM+DEX. Additionally, these 5 patients demonstrated >50% decreases in peripheral blood pDC levels after both tagraxofusp monotherapy and combination therapy. Conclusions: Tagraxofusp was well-tolerated, with a predictable and manageable safety profile, when dosed in combination with POM+DEX in patients with r/r MM. Evidence of pDC suppression in peripheral blood and BM was observed in this patient population. 5 patients that received tagraxofusp and POM+DEX combination had PRs and decreases in pDC levels while on treatment with tagraxofusp. Given CD123 expression on pDCs in the tumor microenvironment and the potential synergy of tagraxofusp with certain MM agents including POM, tagraxofusp may offer a novel mechanism of action in MM. NCT02661022. Disclosures Richardson: Oncopeptides: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding. Gasparetto:Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Zonder:Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Consultancy, Membership on an entity's Board of Directors or advisory committees. Martin:Roche and Juno: Consultancy; Amgen, Sanofi, Seattle Genetics: Research Funding. Chen:Stemline Therapeutics: Employment, Equity Ownership. Brooks:Stemline Therapeutics: Employment, Equity Ownership, Patents & Royalties. McDonald:Stemline Therapeutics: Employment, Equity Ownership. Rupprecht:Stemline Therapeutics: Employment, Equity Ownership. Wysowskyj:Stemline Therapeutics: Employment, Equity Ownership. Chauhan:C4 Therapeutics.: Equity Ownership; Stemline Therapeutics: Consultancy. Anderson:Gilead Sciences: Other: Advisory Board; Janssen: Other: Advisory Board; Sanofi-Aventis: Other: Advisory Board; OncoPep: Other: Scientific founder ; C4 Therapeutics: Other: Scientific founder .

The PI3K Inhibitor GDC-0941 Synergizes with Standard of Care Therapies to Induce Growth Inhibition and Apoptosis of Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3788-3788
Author(s):  
Veerendra Munugalavadla ◽  
Leanne Berry ◽  
Changchun Du ◽  
Sanjeev Mariathasan ◽  
Dion Slaga ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Research Funding ◽  
Single Agent ◽  
Standard Of Care ◽  
Rational Approach ◽  
Current Standard ◽  
Employment Equity ◽  
Specific Flow ◽  
Equity Ownership

Abstract Abstract 3788 Poster Board III-724 Multiple myeloma (MM) is a malignancy characterized by clonal expansion and accumulation of long-lived plasma cells within the bone marrow. Phosphatidylinositol 3' kinase (PI3K) -mediated signaling is frequently dysregulated in cancer and controls fundamental cellular functions such as cell migration, growth, survival and development of drug resistance in many cancers, including MM, and therefore represents an attractive therapeutic target. Here, we demonstrate in vitro, that a potent and selective pan-isoform PI3Kinhibitor, GDC-0941, modulates the expected pharmacodynamic markers, inhibits cell-cycle progression and induces apoptosis; overcomes resistance to apoptosis in MM cells conferred by IGF-1 and IL-6; and is additive or synergistic with current standard of care drugs including dexamethasone, melphalan, lenolidamide and bortezomib. In cell lines we find sensitivity to GDC-0941 is positively correlated with pathway activation as determined by phospho-AKT-specific flow-cytometry and Western-blot analysis. Preliminary results indicate apoptosis of MM cells is correlated with increased expression of the proapoptotic BH3-only protein BIM; mechanisms of increased apoptosis in MM will be further explored and an update presented. We further extend these in vitro findings to show that GDC-0941 has activity as a single agent in vivo and combines well with standard of care agents in several murine xenograft models to delay tumor progression and prolong survival. Our results suggest that GDC-0941 may combine well with existing therapies, providing a framework for the clinical use of this agent, and a rational approach to improving the efficacy of myeloma treatment. Disclosures: Munugalavadla: Genentech: Employment, Patents & Royalties. Berry:Genentech: Employment, Patents & Royalties. Du:Genentech, Inc.: Employment, Equity Ownership. Mariathasan:Genentech: Employment, Patents & Royalties. Slaga:Genentech: Employment, Patents & Royalties. Sun:Genentech Inc.: Employment. Chesi:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Bergsagel:Genentech: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

QT Interval Effects and M-Protein Response in a Phase 1 Study of Siltuximab (Anti-IL-6 Monoclonal Antibody) in Patients with Monoclonal Gammopathy of Undetermined Significance, Smoldering Multiple Myeloma, or Indolent Multiple Myeloma.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2972-2972
Author(s):  
Sheeba K. Thomas ◽  
Alexander Suvorov ◽  
Lucien Noens ◽  
Oleg Rukavitsin ◽  
Joseph Fay ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Studies ◽  
Qt Interval ◽  
Research Funding ◽  
Phase 1 ◽  
Single Agent ◽  
M Protein ◽  
Qtc Interval ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 2972 Introduction Siltuximab is a chimeric monoclonal antibody that binds human interleukin (IL)-6 with high affinity. Formal assessments of siltuximab's effects on cardiac repolarization using triplicate electrocardiograms (ECGs) have not yet been performed in clinical studies. A phase 1 study was conducted to evaluate the effect of siltuximab, administered at the highest dose level used in clinical studies, on the QT interval in patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), or indolent multiple myeloma (IMM, i.e., asymptomatic MM with ≤3 lytic bone lesions but no other end organ damage). Methods Thirty patients with MGUS, SMM, or IMM who met the following criteria on ECG at screening: pulse 45−90 bpm, QTcF and QTcB ≤500 ms, QRS <100 ms, and PR <200 ms received siltuximab 15 mg/kg q3w as a 60 min IV infusion for 4 cycles. Patients were excluded for significant cardiac disease. ECGs and pharmacokinetics assessment were performed at Cycle 1 (pre-infusion [baseline]; end of infusion; and 1, 3, and 24 hrs post-infusion) and at Cycle 4 (pre-infusion, end of infusion, and 1 hr post-infusion). At all timepoints, triplicate 12-lead ECGs were conducted and evaluated by a central cardiology laboratory. No effect on QTc interval was concluded if the upper limit of the least square (LS) mean 90% confidence interval (CI) for the change from baseline QTc at each time point was <20 ms. Safety data were also collected. Preliminary assessment of clinical activity was performed using M-protein measurements from local laboratories. Patients achieving a 50% reduction from baseline in M-protein after 4 cycles were eligible for extended siltuximab therapy (15 mg/kg q4w). Results Thirty patients (14 MGUS, 15 SMM, 1 IMM) with median age 59.5 (range 24, 79) yrs were enrolled. Median serum protein electrophoresis was 1.21 (range 0, 5.4) g/dL, and median urine protein electrophoresis was 0 (range 0, 267) mg/24 hrs. Twenty-eight patients completed all 4 treatment cycles, among whom 27 were evaluable for the primary endpoint of QT interval assessment. The maximum mean increase in QTc from baseline occurred 3 hrs after the Cycle 1 infusion (QTcF = 3.2 ms [LS mean 90% CI −0.01, 6.45]; QTcB = 2.7 ms [LS mean 90% CI −0.69, 6.14]). At all other time points for both QTcF and QTcB, the mean increase from baseline was ≤1.5 ms and the upper limit of LS mean 90% CI was ≤5.07 ms. An effect of siltuximab on QTc interval was therefore ruled out. Furthermore, no patient had a QTcF or QTcB increase from baseline >30 ms, and no correlation was observed between siltuximab serum concentrations and change from baseline in QTcB or QTcF. Twenty (67%) of 30 treated patients had ≥1 adverse events (AEs). AEs reported by ≥10% of patients were nausea, fatigue (20% each); thrombocytopenia, headache (each 13%); dyspnea, leukopenia, neutropenia, paraesthesia, and upper respiratory tract infection (each 10%). The majority of AEs were grade ≤2. However, 8 (27%) patients had ≥1 AE grade ≥3: neutropenia (n=3); hypertriglyceridemia, hypertension, hypotension, leukopenia, and myalgia (each n=1); and 1 patient had grade 3 ascites, cellulitis, peripheral edema, portal hypertension, and hepatic cirrhosis (diagnosis made during hospitalization). This patient discontinued treatment due to cellulitis (possibly related to siltuximab) with peripheral edema and ascites (not related to siltuximab). A second patient discontinued treatment due to grade 2 atrial fibrillation that was not related to siltuximab. No severe infusion reactions or deaths were reported. After the first 4 cycles (3 mos), 3 MGUS patients achieved an M-protein response (≥50% reduction from baseline), and 9 patients (3 MGUS, 5 SMM, 1 IMM) had minor responses (≥25% and <50% reduction from baseline). Two patients who qualified for extended treatment with siltuximab continued to receive therapy (17 and 6 cycles, respectively) at the time of database lock. Conclusion Siltuximab, given at the highest dose level used in clinical studies, did not affect the QTc interval. Overall safety was similar to what has been previously reported for other single-agent siltuximab studies. M-protein responses were seen by local laboratory assessment within the first 4 cycles. A randomized phase 2 study is ongoing to further evaluate the efficacy and safety of single-agent siltuximab in high-risk SMM. Disclosures: Thomas: Millenium: Research Funding; Novartis: Research Funding; Immunomedics: Research Funding; Johnson & Johnson: Research Funding; Celgene: Research Funding; Onyx: Membership on an entity's Board of Directors or advisory committees. van de Velde:Johnson & Johnson: Employment, Equity Ownership. Bandekar:Johnson & Johnson: Employment, Equity Ownership. Puchalski:Johnson & Johnson: Employment, Equity Ownership. Qi:Johnson & Johnson: Employment, Equity Ownership. Uhlar:Johnson & Johnson: Employment, Equity Ownership.

Glutaminase Inhibitor CB-839 Synergizes with Pomalidomide in Preclinical Multiple Myeloma Models

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4720-4720 ◽  
Author(s):  
Francesco Parlati ◽  
Mathew Gross ◽  
Julie Janes ◽  
Evan Lewis ◽  
Andy MacKinnon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Clinical Trials ◽  
Growth Inhibition ◽  
Tumor Cells ◽  
Single Agent ◽  
Aspartate Transcarbamylase ◽  
Employment Equity ◽  
Two Agents ◽  
Equity Ownership ◽  
Hematological Tumor

Abstract Many hematological tumor cells are dependent on glutamine for growth and survival. Glutamine is the most abundant amino acid in plasma and can be utilized by tumor cells for production of energy and generation of building blocks for the synthesis of macromolecules. Small molecule CB-839 inhibits glutaminase (GLS) activity thereby blocking cellular glutamine utilization resulting in an anti-tumor effect in several hematological tumor types including multiple myeloma (MM), acute lymphocytic leukemia, and several types of non-Hodgkin’s lymphoma [Parlati et al. Blood 2013 122:4226]. Phase 1 clinical trials have been initiated to test the safety, pharmacokinetics, pharmacodynamics, and clinical activity of single agent CB-839 in several hematological malignancies. In anticipation of potential combinations of CB-839 with standard of care agents in future MM clinical trials, we tested the effects of CB-839 in combination with the IMiD, pomalidomide (POM). POM caused complete growth inhibition in MM.1S cells with an EC50 of 16 nM as opposed to partial growth inhibition in RPMI8226 cells, with an EC50 of 130 nM. CB-839 caused complete growth inhibition in MM.1S cells with an EC50 value of 26 nM and produced a cytotoxic effect in RPMI8226 cells with an EC50 of 160 nM. When combined, CB-839 enhanced the anti-proliferative activity of POM in both POM-sensitive MM.1S and POM-resistant RPMI8226 cells resulting in a synergistic anti-tumor effect as demonstrated by combination index values between 0.18-0.62 (mean= 0.36) for the MM.1S and 0.25-0.72 (mean= 0.38) for the RPMI8226 cells. To investigate the mechanism that underlies the observed synergy, RPMI8226 cells were treated for 24 hours and changes in proteins and metabolites were measured by reverse-phase-protein array and LC/MS, respectively. When treated with CB-839 alone, RPMI8226 cells respond by decreasing mTOR pathway signaling proteins (e.g. phospho-mTOR, phospho-p70S6K, phospho-PRAS40, phospho-S6), decreasing the amount of oncogenic proteins (c-Myc and c-Kit), and increasing programmed cell death pathway proteins (e.g. cleaved caspase 7, cleaved PARP), consistent with the cytotoxic activity observed for CB-839. Several of these changes were further enhanced in the presence of POM (e.g. phospho-p70S6K, phospho-S6, phospho-PRAS40, c-kit, c-Myc), however only the enhanced decrease in c-Myc reached statistical significance. Metabolite analysis showed changes with CB-839 consistent with GLS inhibition (e.g. decreases in glutamate, aspartate, succinate and malate and increases in glutamine). On the other hand, single agent POM caused very modest changes in the metabolite profile. When the two agents were combined, metabolite levels were consistent with those observed with single agent CB-839, with the notable exception of carbamoyl-aspartate where lower levels were measured in the combination group in comparison to cells treated with either agent alone. Carbamoyl-aspartate is an intermediate in the pyrimidine biosynthesis pathway and is synthesized by the multi-catalytic enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase), an enzyme that is regulated by mTOR [Ben-Sahra et al. (2013) Science339: 1323-8]. These observations suggest that CB-839 dampens mTOR signaling and POM may further attenuate this response, possibly contributing to the synergistic anti-tumor effect. These data motivated testing the anti-tumor effect of the combination of CB-839 and POM in mice bearing RPMI8226 xenografts. Oral dosing with single agent CB-839 and POM resulted in tumor growth inhibition (TGI) of 64% and 46%, respectively, whereas the combination of the two agents resulted in a TGI of 97%. Efficacious doses of CB-839 and POM alone or in combination were well tolerated with no effect on animal body weight. These promising results indicate that GLS inhibition with CB-839 in combination with POM may provide therapeutic benefit in MM and provide motivation for future clinical studies. Disclosures Parlati: Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Lewis:Calithera Biosciences: Employment, Equity Ownership. MacKinnon:Calithera Biosciences: Employment, Equity Ownership. Rodriguez:Calithera Biosciences: Employment, Equity Ownership. Shwonek:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership.

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