Triphase Accelerator Research Articles

Exploring Alternative Dosing Regimens of Single-Agent Belantamab Mafodotin on Safety and Efficacy in Patients with Relapsed or Refractory Multiple Myeloma: DREAMM-14

Exploring Alternative Dosing Regimens of Single-Agent Belantamab Mafodotin on Safety and Efficacy in Patients with Relapsed or Refractory Multiple Myeloma: DREAMM-14

Safety and Efficacy of Cusatuzumab in Combination with Venetoclax and Azacitidine (CVA) in Patients with Previously Untreated Acute Myeloid Leukemia (AML) Who Are Not Eligible for Intensive Chemotherapy; An Open-Label, Multicenter, Phase 1b Study

Safety and Efficacy of Cusatuzumab in Combination with Venetoclax and Azacitidine (CVA) in Patients with Previously Untreated Acute Myeloid Leukemia (AML) Who Are Not Eligible for Intensive Chemotherapy; An Open-Label, Multicenter, Phase 1b Study

Trph-222, a Novel Anti-CD22 Antibody Drug Conjugate (ADC), Has Signficant Anti-Tumor Activity in NHL Xenografts and Is Well Tolerated in Non-Human Primates

CD22 is a B-cell specific transmembrane glycoprotein belonging to the SIGLEC family of lectins. Its expression is restricted to B lineage cells from the early immature/pre-B-cell stage through the mature naïve stage, and is downregulated on the surface of plasma cells. CD22 is highly expressed on a wide range of malignancies arising from mature B-cells, including NHL (non-Hodgkin's lymphoma), DLBCL (diffuse large B cell lymphoma), MCL (mantle cell lymphoma), MZL, B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and small lymphocytic leukemia. The restricted lineage of CD22 and rapid internalization upon antibody binding make CD22 an appealing target for an antibody-drug conjugate (ADC) therapeutic for B cell malignancies. Prior attempts at targeting CD22 using ADC technology have been limited clinically in NHL due to an unacceptable or poor therapeutic index. Subsequently the ADC field has focused on more stable and site specific conjugation of payloads to increase the therapeutic index of ADCs.TRPH-222 is an ADC comprised of an anti-CD22 antibody site-specifically modified at one site in each heavy chain to express formylglycine (FG), allowing for site-specific conjugation of a maytansinoid payload, a protease-insensitive spacer, and a functional group for coupling to an aldehyde on the antibody FG residues. This SMARTAG™ site specific non-cleavable conjugation enforces a maximum drug to antibody ratio (DAR) of 2.0.The aim of this study was to characterize the anti-tumor activity of TRPH-222 in mouse xenografts and tolerability in non-human primates (NHP).In the WSU-DLCL2 xenograft model, once weekly intravenous (IV) dosing of 1, 3 or 10mg/kg or TRPH-222 reduced tumor volume by 51%, 100% and 100% after 28 days. A once every three-week dose of 10mg/kg reduced tumor volume by 100% from pre-dosing measurements. In contrast, rituximab (30mg/kg, weekly) resulted in 55% tumor growth inhibition. In both SU-DHL-2 (DLBCL) and Granta-519 (MCL) xenograft models, weekly dosing at 10mg/kg resulted in tumor stasis.The tolerability and PK of TRPH-222 were evaluated in cynomolgus monkeys. Animals were dosed IV once every 3 weeks (the anticipated clinical dosing schedule) for either 2 (5, 12.5, or 25 mg/kg) or 3 (50 mg/kg) doses. TRPH-222 was well tolerated at all doses evaluated and all animals survived to study termination, with no TRPH-222-related effects on clinical observations or body weight. Aspartate aminotransferase (AST) activity was mildly increased in animals at ≥12.5 mg/kg/dose, while minimal increases in alanine aminotransferase (ALT) activity were noted at 50 mg/kg/dose. Minor microscopic findings of Kupffer cell hypertrophy/hyperplasia were noted in the livers of monkeys treated at 50 mg/kg IV; this finding was not considered adverse based on minimal severity and lack of degeneration/necrosis. Preliminary PK data demonstrate the stability of TRPH-222, as the ratio of the area under the curve (AUC) from day 0-42 for the ADC was ~76-88% that of the antibody backbone suggesting that the ADC has a long half-life and that the linker is stable in vivo.In conclusion, the studies reported here demonstrate that TRPH-222 has significant anti-tumor activity in a range of B-cell lymphoma models and that very high doses of TRPH-222 are well tolerated in monkeys. IND-enabling safety studies are ongoing to support clinical evaluation of TRPH-222 in 2018. These data support the argument that TRPH-222 will have a large therapeutic window due to the stability of conjugation of the maytansinoid payload to the anti-CD22 antibody; doses tolerated in the monkey study described above correspond to human equivalent doses significantly higher than for most ADCs approved or currently under clinical evaluation. To our knowledge, this is the highest dose of a maytansine containing ADC that has been tolerated in monkeys and raises the potential for a best in class therapy for NHL in addition to a best in class ADC technology. DisclosuresMaclaren:Triphase Accelerator: Employment. Levin:Triphase Accelerator: Employment. Lowman:Triphase Accelerator: Employment. Trikha:Triphase Accelerator: Employment; Encycle Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Targeting Proteasome- Associated Ubiquitin Receptor PSMD4/Rpn10 in Multiple Myeloma

The LeBow Institute for Myeloma Therapeutics and Jerome Lipper Myeloma Center, Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MABackground and Rationale Normal cellular homeostasis is maintained by a balanced regulation of protein synthesis and degradation via the ubiquitin proteasome system (UPS). Deregulation of the UPS is linked to pathogenesis of various human diseases, including cancer. Targeting the 20S proteasome in the UPS via proteasome inhibitors bortezomib, carfilzomib, or ixazomib has proven effective therapy for relapsed, and newly diagnosed multiple myeloma (MM); however, dose-limiting toxicities and the development of resistance limit their long-term utility. Therapeutic strategies directed against ubiquitin receptors upstream of the 20S proteasome may allow for more specific targeting of the UPS, with fewer off-target activities and toxicities. Ubiquitin-tagged proteins are recognized by 19S proteasome-associated ubiquitin receptors; the ubiquitin moiety is then removed from the substrate protein via deubiquitylating (DUB) enzyme enzymatic activity; target protein is then unfolded by the AAA-ATPases; followed by degradation of proteins through 20S proteolytic activities. Here we examined the role of ubiquitin receptor Rpn10/PSMD4 in MM using RNA interference strategies.Materials and Methods We utilized MM cell lines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors. MM.1S cells were transiently transfected with control short interfering RNA (siRNA) or Rpn10-siRNA ON TARGET plus SMART pool using the cell line Nucleofector Kit V. Signal transduction pathways were evaluated using immunoblotting. Cell viability were assessed using WST-1 assay. Proteasome activity was measured as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). Statistical significance of data was determined using a Student's t test.Results1) A statistically significant inverse correlation was noted between Rpn10 levels and overall patient survival (n=175) (p= 0.00064). 2) Analysis of PSMD4/Rpn10 gene expression showed a significantly higher level in primary patient MM cells versus normal plasma cells or PBMCs (p = 0.023 or p = 0.001, respectively). 3) Rpn10 protein is more highly expressed in MM cells than in normal cells. 4) Functional studies showed that blockade of Rpn10 using RNA interference (siRNA and shRNA) significantly decreased MM cell viability (p < 0.001; n=3). 5) Rpn10-shRNA knockdown decreased MM cell proliferation. These findings were further confirmed in doxycycline-inducible Rpn10-shRNA knockout cells. 6) Transfection of Rpn10-siRNA in MM cells increased accumulation of poly-ubiquitylated proteins. 7) To test whether Rpn10 knockdown blocks cellular proteasome function, we used a reporter cell line expressing ubiquitin-tagged GFP that is constitutively targeted for proteasomal degradation. Rpn10-siRNA increased accumulation of the Ub-GFP reporter indicating impaired proteasome degradation. 8) Mechanistic studies show that Rpn10-siRNA-triggered MM cell death is associated with: (i) cell cycle arrest and apoptosis; (ii) increase in poly-ubiquitylated proteins; and (iii) activation of caspases and induction of endoplasmic stress response signaling.Conclusion Our preclinical data validates targeting 19S proteasome ubiquitin receptor Rpn10 upstream of proteasome in the ubiquitin proteasomal cascade, and provides the framework for development of proof-of-concept inhibitors targeting Rpn10 for future evaluation in clinical trials. DisclosuresChauhan:Triphase accelerator: Consultancy. Anderson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Oncopep: Other: scientific founder.

A Phase I Pharmacokinetic (PK) and Safety Study of Trph-222 in Patients with Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma (R/R NHL): Dose-Escalation Results

A Phase I Pharmacokinetic (PK) and Safety Study of Trph-222 in Patients with Relapsed/Refractory B-Cell Non-Hodgkin Lymphoma (R/R NHL): Dose-Escalation Results

Trials in Progress: A Phase I Study to Evaluate the Safety and Pharmacokinetic Profiles of CB-5339 in Participants with Relapsed/Refractory Acute Myeloid Leukemia or Relapsed/Refractory Intermediate or High-Risk Myelodysplastic Syndrome

Trials in Progress: A Phase I Study to Evaluate the Safety and Pharmacokinetic Profiles of CB-5339 in Participants with Relapsed/Refractory Acute Myeloid Leukemia or Relapsed/Refractory Intermediate or High-Risk Myelodysplastic Syndrome

Real-World Incidence and Clinical and Economic Burden of Managing Ocular Events in Patients with Active Multiple Myeloma

Introduction: Some novel cancer and supportive care therapies, such as chemotherapy, steroids, and immunotherapies, are known to cause eye-related side effects such as dryness or altered vision (Fu et al Oncotarget 2017). However, there is a paucity of evidence documenting their disease burden in practice. The aim of this retrospective, cohort study was to assess the incidence, and clinical and economic burden of managing ocular events (OEs) in patients receiving multiple myeloma (MM) treatment in clinical practice. Methods: A cohort of adult patients with MM was identified from a large U.S. insurance claims database (IQVIA PharMetrics® Plus database), which is sourced from commercial insurance companies. Incidence, outpatient care, emergency room (ER) visits, hospitalizations, and costs were assessed for 5 OEs of interest: keratopathy/keratitis, blurred vision/decreased acuity, dry eye, eye pain, and photophobia. These OEs were identified by cross-walking from MedDRA 'corneal disorders' codes to international classification of disease (ICD) codes, which were confirmed by clinical experts. Incidence of OEs was assessed as the proportion of patients with OE after MM treatment initiation who did not have diagnosis indicative of OE in the 12-month baseline period. Descriptive analyses were performed, and resource utilization and costs (in US dollars) were reported on a per-patient-per-month (PPPM) basis. A separate cohort of hematology patients (N=26,923) was identified from the same database. The results were benchmarked against this larger cohort of patients. Results: Of the 2120 patients with incident MM (mean [SD] age: 60.2 [9.8]) included in the cohort, 248 (11.7%) patients had at least 1 incident OE during an average of 29.8 months of follow-up after initial MM treatment. Proportion of patients with at least 1 incident OE was 7.4% in the benchmark hematology cohort. Dry eye (n=129, 6.1%), blurred vision/decreased acuity (n=73, 3.4%), and keratopathy/keratitis (n=52, 2.5%) were the most frequent OEs observed. The incidence remained largely constant across baseline demographic characteristics, comorbid conditions, and lines of MM therapies. Most incident OEs were diagnosed in the outpatient (OP)/physician's office setting by ophthalmologists (69.5%), except for blurred vision, which was predominantly diagnosed in non-ophthalmologist/non-optometrist setting. Median PPPM costs for managing OEs was $27, which is &amp;lt;1% of median all-cause costs during the follow-up period (median total all-cause PPPM costs: $17,286; median MM-related PPPM costs: $13,851). These costs were predominantly contributed by OP care (median PPPM cost: $19), including prescriptions (median PPPM cost: $16). ER visits (median PPPM cost: $66) and inpatient visits (median PPPM cost: $16) were uncommon (Table). Median PPPM costs for managing these OEs in hematology cohort was $20. Conclusions: Incident OEs were observed in ~12% of patients receiving treatment for MM based on real-world data, with the most common OEs being keratopathy/keratitis, dry eye, and blurred vision. When evaluated from the perspective of median PPPM costs, the clinical and economic burden for treating OEs was very low when compared with total all-cause PPPM costs or MM-related PPPM costs. The vast majority of these OEs require only OP care. ER visits or inpatient care were extremely rare. Further real-world evidence is warranted to assess the relationship between OEs and MM treatment. Funding: GSK (study: 208295). Table. Median OE-related PPPM Costs (US Dollars) in Incident MM Cohort (N=2,120) Table Disclosures Wang: GSK: Current Employment, Current equity holder in publicly-traded company. Ferrante:GSK: Current Employment, Current equity holder in publicly-traded company. Maiese:GSK: Current Employment, Current equity holder in publicly-traded company. Willson:GSK: Current Employment, Current equity holder in publicly-traded company. Chen:GSK: Consultancy, Research Funding. Nunna:GSK: Consultancy, Research Funding. Sun:GSK: Consultancy, Research Funding. Kleinman:GSK: Consultancy; Eyeon Therapeutics, LLC.: Current equity holder in private company; Triphase Accelerator U.S Corporation: Consultancy; ONL Therapeutics, Inc: Consultancy; Revolution Medicines, Inc: Consultancy; Editas Medicine, Inc: Consultancy; Cleave Therapeutics, Inc: Consultancy; Coherus Biosciences, Inc: Consultancy; Synergy Research Inc: Consultancy; Zenith Epigenetics Ltd: Consultancy. Sansbury:GSK: Current Employment, Current equity holder in publicly-traded company.

DREAMM-2: Single-Agent Belantamab Mafodotin (Belamaf) Effects on Patient-Reported Outcome (PRO) Measures in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

DREAMM-2: Single-Agent Belantamab Mafodotin (Belamaf) Effects on Patient-Reported Outcome (PRO) Measures in Patients with Relapsed/Refractory Multiple Myeloma (RRMM)

Celgene accesses WDR5-targeted compound

Celgene will pay $40 million up front for an option to acquire Triphase Accelerator’s small molecule targeting WDR5 protein, a key player in the formation of protein complexes that indirectly modify genes. Leukemia and other blood cancers can arise due to the misregulation of WDR5-associated protein complexes. The deal for the preclinical compound is Celgene’s third cancer pact since Bristol-Myers Squibb announced it would buy the New Jersey–based company for $74 billion last month.

Pmd-107: Marizomib, Pomalidomide and Low Dose-Dexamethasone Combination Study in Relapsed/Refractory Multiple Myeloma (NCT02103335): Full Enrollment Results from a Phase-1 Multicenter, Open Label Study

Marizomib (MRZ) is a novel, irreversible, pan subunit proteasome inhibitor (PI) with preclinical evidence demonstrating in vitro and in vivo activity in multiple myeloma (MM). This study was designed to evaluate the safety and antimyeloma activity of pomalidomide (POM), MRZ and low dose dexamethasone (Lo-DEX) (PMD) in patients with relapsed and refractory multiple myeloma (RRMM).Thirty-eight heavily pretreated patients with RRMM were enrolled [dose-escalation cohort (n=14); recommended Phase 2 dose (RP2D) cohort (n=24)]. IV MRZ (0.3 to 0.5 mg/m2) was administered on Days (D) 1, 4, 8, 11; POM (3 or 4 mg) on D1 through 21; and Lo-DEX (5 or 10 mg) on D1, 2, 4, 5, 8, 9, 11, 12, 15, 16, 22, 23 of every 28-D cycle. Patients received a median of 4 (range 1-9) prior lines of therapy; 100% received prior lenalidomide (LEN) and bortezomib (BTZ), 34% carfilzomib (CFZ), and 50% thalidomide. 53% of patients were refractory to both LEN and BTZ and 21% were refractory to LEN, BTZ, and CFZ.There were no dose limiting toxicities during the study. The most common study treatment related ≥Grade 3 adverse events (AEs) were neutropenia (11/38 pts: 29%), pneumonia (4/38 pts 11%), anemia (4/38 pts; 11%), thrombocytopenia (4/38 pts; 11%), and febrile neutropenia (2/38 pts; 5%), with two grade 4 AEs (neutropenia related to POM and viral infection related to DEX), and one grade 5 AE (cardio-respiratory arrest from a suspected PE related to POM). Overall, MRZ was well tolerated, did not add to the incidence or severity of POM/Lo-DEX AEs and the regimen may have fewer hematological and infectious AEs compared to that observed with POM/Lo-DEX.MRZ pharmacokinetic analysis revealed that it was rapidly cleared with a short T1/2 (6.2-11mins) and a large volume of distribution (41-86L) suggesting extensive tissue distribution. Pharmacodynamic analysis demonstrated rapid and robust inhibition of chymotrypsin-like activity in both packed whole blood (PWB) and peripheral blood mononuclear cells (PMBCs), reflecting the irreversible binding nature of MRZ. Evolving inhibition of trypsin-like and caspase-like proteasome activity was also observed in PWB and PBMC with continued dosing.The overall response rate (ORR) and clinical benefit rate (CBR) for the 36 response evaluable patients was 53% (19/36) and 64% (23/36), respectively (Table 1). Subpopulation analysis demonstrated an ORR of 50% (5/10) in high risk cytogenetic patients, 56% (10/18) in LEN/BTZ refractory patients, 71% (5/7) in LEN/BTZ/CFZ refractory patients and 80% (8/10) in CFZ refractory patients. These data compare favorably against POM/Lo-Dex with a near doubling of ORR in both the total patient population and the double refractory patients. Substantial activity in high-risk patients that are triple refractory and in patients that are refractory to CFZ in prior last regimen was observed. MRZ activity in RRMM patients exposed and/or refractory to multiple PIs is likely a consequence of its unique pan proteasome subunit inhibitory actions. In conclusion, MRZ in combination with POM and Lo-DEX was well tolerated and demonstrated promising activity in heavily pretreated, high-risk RRMM patients. [Display omitted] DisclosuresHarrison:Janssen-Cilag: Research Funding, Speakers Bureau; Celgene: Honoraria. Zonder:Prothena: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Pharmacyclics: Other: DSMC membership. Khot:Amgen: Honoraria; Janssen: Consultancy; Pfizer: Speakers Bureau. Anderson:C4 Therapeutics: Equity Ownership; Millennuim: Membership on an entity's Board of Directors or advisory committees; Oncoprep: Equity Ownership; C4 Therapeutics: Equity Ownership; Gilead: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Acetylon: Equity Ownership; Acetylon: Equity Ownership; Oncoprep: Equity Ownership; Millennuim: 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; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. MacLaren:Triphase Accelerator: Employment, Equity Ownership. Reich:Triphase Accelerator: Consultancy. Trikha:Encycle Therapeutics: Consultancy, Equity Ownership; Triphase Accelerator: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Marizomib for CNS-Multiple Myeloma

Marizomib, a natural marine product, is an irreversible proteasome inhibitor (PI) that has distinct advantages over the currently approved PIs in that it irreversibly inhibits all three enzymatic activities of the proteasome and has been demonstrated to cross the blood brain barrier. Several studies have demonstrated that marizomib (MRZ) penetrates and can be retained in the CNS and that it substantially inhibits proteasome activity in the brain; radiolabeled MRZ showed 30% CNS biodistribution compared with blood levels in rats; it elicits a significant anti-tumor effect in a rodent model of malignant glioma; and pharmacological inhibition of proteasome activity has been observed in primate brains. MRZ is currently under clinical investigation in relapsed-refractory multiple myeloma and grade IV malignant glioma. CNS-multiple myeloma (MM) is a rare manifestation of extra-medullary disease with very limited therapeutic options. Its prevalence is increasing as anti-myeloma therapies become more effective at treating systemic disease, resulting in the CNS becoming a sanctuary site for the disease further highlighting the urgent unmet clinical need in this patient population. MRZ has demonstrated encouraging activity in non-CNS-MM, and has emerging clinical activity in glioma making it a potentially promising CNS-MM therapeutic intervention.To date 3 patients have been treated under compassionate use protocol with MRZ for CNS-involved MM. Two of these patients have received sufficient doses of MRZ in combination with DEX to evaluate its activity in this indication. MRZ was dosed initially at 0.5mg/m2 and then increased to 0.7mg/m2once tolerability had been established on the schedule currently in use on the glioma study (days 1, 8, and 15 of a 28 day cycle as a 10 minute infusion). Both patients, who had received prior cranio-spinal radiotherapy and intrathecal chemotherapy with no response, have tolerated MRZ well with no unexpected adverse events. The first patient had an 89% reduction in cerebrospinal fluid (CSF) plasmacytosis and 77% serum LDH reduction. After 5 months daratumumab was added to MRZ and further clinical improvement was observed (resolution of double vision and becoming fully ambulatory). Six months after MRZ initiation the patient progressed clinically and stopped treatment. Clinical symptoms disappeared in the second patient in the second month of MRZ with complete resolution of CSF plasmacytosis and CSF monoclonal spike. The patient is currently undergoing the 4th cycle of treatment with sustained resolution of saddle anesthesia and no further signs of cauda equina symptoms. Collectively, these cases provide preliminary evidence that MRZ may have utility as a therapeutic for CNS-MM and also demonstrate that combination of MRZ and daratumumab may provide benefit in this indication. Follow up is ongoing on the second patient and more mature data will be presented on this patient and any additional patients subsequently treated under compassionate use for CNS-MM. DisclosuresMacLaren:Triphase Accelerator: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Trikha:Encycle Therapeutics: Consultancy, Equity Ownership; Triphase Accelerator: Employment, Equity Ownership. Parameswaran:Takeda: Consultancy.

Celgene acquires a myeloma treatment

Celgene has acquired the proteasome inhibitor marizomib from Triphase Accelerator for an undisclosed sum plus future milestone payments. Triphase has completed Phase I clinical trials of marizomib as a treatment for glioblastoma and multiple myeloma. Celgene already markets the multiple myeloma treatments Revlimid and Thalomid. Triphase, a spin-off from the Ontario Institute for Cancer Research, granted Celgene the option to buy the drug in 2014.

Oral Proteasome Inhibitor Marizomib and IMiD® Imunomodulatory Drug Pomalidomide Trigger Synergistic Anti-Myeloma Activity and Enhanced Proteasome Inhibition in Vitro and In Vivo

Introduction Proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity and drug resistance. A novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities (Chauhan et al., Cancer Cell 2005, 8:407-419). Pomalidomide is an analogue of thalidomide with potent immunomodulatory activity. Based on increased progression-free survival, pomalidomide has been approved by the FDA for the treatment of patients with RRMM who have received at least two prior therapies, including lenalidomide and bortezomib, and who showed disease progression on or within 60 days of completion of the most recent therapy. Here we utilized in vitro and in vivo models of MM to examine the anti-MM activity of combined marizomib and pomalidomide. Animal model studies examined the efficacy of marizomib (PO) using both single weekly and twice weekly schedule either alone or together with pomalidomide (PO).Methods Cell viability, and apoptosis were performed using WST/MTT, and Annexin V, respectively. Synergistic anti-MM activity was determined with CalcuSyn software program. Proteasome activity was measured, as in prior study (Chauhan et al., Cancer Cell 2005). MM.1S-tumor-bearing mice were treated with vehicle control, marizomib (PO) pomalidomide (PO), or marizomib plus pomalidomide at the indicated doses for 21 days on a twice-weekly or once weekly schedule for marizomib and 4 consecutive days weekly for pomalidomide. Statistical significance was determined using a Student's t test. Pomalidomide was purchased from Selleck chemicals; and marizomib was obtained from Triphase Accelerator, USA.Results MM cell lines (MM.1R, MM.1S, INA-6, RPMI-8226, LR5, Dox-40, bortezomib-sensitive ANBL6.WT, and bortezomib-resistant ANBL6.BR) and primary patient MM cells were pretreated with pomalidomide for 24h; marizomib was then added for an additional 24h, followed by measurement of cell viability. A significant decrease in viability of all cell lines was observed in response to treatment with combined low doses of marizomib and pomalidomide vs. either agent alone. Isobologram analysis confirmed the synergistic anti-MM activity of these agents (CI < 1.0). The cytotoxicity of combination therapy was observed in MM cell lines sensitive and resistant to novel therapies, and in p53-null ARP-1 MM cells. A significant decrease in cell viability of all patient MM cells was noted after combination therapy as compared to either compound alone (p < 0.05 for all patients). In contrast, combined low doses of marizomib plus pomalidomide did not significantly affect the viability of normal PBMCs, suggesting a favorable therapeutic index for this combination regimen. Tumor cells from 5 of 7 patients were obtained from patients whose disease was progressing while on bortezomib, dex, and lenalidomide therapies. Marizomib plus pomalidomide-induced apoptosis was associated with: 1) activation of caspase-8, caspase-9, caspase-3, and PARP cleavage; 2) downregulation of cereblon (CRBN), IRF4, c-Myc, and Mcl-1; and 3) suppression of CT-L, C-L, and T-L proteasome activities. CRBN-siRNA attenuated marizomib plus pomalidomide-induced MM cells death. Furthermore, marizomib plus pomalidomide inhibited the migration of MM cells and tumor-associated angiogenesis, and overcame cytoprotective effects of BM milieu. Human MM xenograft model study showed that combined low doses of marizomib (twice weekly; PO)) and pomalidomide (4 consecutive days weekly; PO) for 3 weeks were well tolerated, inhibited tumor growth, and prolonged survival. Importantly, combination of marizomib on once weekly (PO) schedule with pomalidomide (PO) was active and led to prolongation of survival. Finally, inhibition of CT-L (63%), T-L (40%) and C-L (29%) proteasome activity was observed in tumors from marizomib plus pomalidomide-treated mice vs. untreated mice.Conclusion Our preclinical data from in vitro studies and in vivo MM xenograftmodels demonstrate that oral marizomib plus pomalidomide trigger synergistic anti-MM activity, enhance proteasome inhibition, and overcome drug resistance. These studies support the continuation of clinical trials of combined marizomib and pomalidomide to improve outcome in patients with RRMM. DisclosuresTrikha:Triphase Accelerator Corporation: Employment. Chauhan:Stemline Therapeutics: Consultancy.

Phase 1, Multicenter, Open-Label, Combination Study (NPI-0052-107; NCT02103335) of Pomalidomide (POM), Marizomib (MRZ, NPI-0052), and Low-Dose Dexamethasone (LD-DEX) in Patients with Relapsed and Refractory Multiple Myeloma

Phase 1, Multicenter, Open-Label, Combination Study (NPI-0052-107; NCT02103335) of Pomalidomide (POM), Marizomib (MRZ, NPI-0052), and Low-Dose Dexamethasone (LD-DEX) in Patients with Relapsed and Refractory Multiple Myeloma

Marizomib Overcomes Compensatory Hyperactivation of Trypsin-like and Caspase-like Subunits to Provide Pan-Proteasome Subunit Inhibition in Patients with Multiple Myeloma and Solid Tumors

Background: Proteasome inhibitors (PIs) are a highly active drug class in multiple myeloma (MM), but development of resistance is commonly observed. Although all clinical-stage PIs are effective inhibitors of chymotrypsin-like (CT-L) proteasome subunit activity, a possible mechanism of resistance is compensatory hyperactivation of the caspase-like (C-L) and trypsin-like (T-L) subunits. Marizomib (MRZ) is a novel, irreversible, second-generation PI under development for the treatment of MM and malignant glioma. MRZ potently inhibits the 3 subunits of the 20S proteasome with a specificity and activity distinct from that of bortezomib and carfilzomib and their oral analogs ixazomib and oprozomib.Methods: In the clinical study NPI-0052-102, we evaluated the pharmacodynamic (PD) activity of MRZ against all three proteasome subunits in whole blood after single, or repeated administration, via two schedules in patients (pts) with previously treated advanced malignancies (solid tumors or hematologic). Pts received MRZ via Schedule A - weekly dosing for 3 doses in 4-week cycles by IV infusion over 1 to 10 min, or Schedule B - twice-weekly dosing for 4 doses in 3-week cycles by IV infusion over 10 min to 2 hr. Blood samples were collected on Days 1 and 15 (Schedule A) or 1 and 11 (Schedule B), pelleted by centrifugation and frozen within 48 hrs. Pellets were lysed and the activity of all 3 proteasomal subunits was assayed using specific fluorogenic substrates.Results: Partial or complete inhibition of all three proteasome subunits was achieved with both once-weekly and twice-weekly MRZ dosing. The rank order of sensitivity, CT-L > T-L > C-L, was as expected from the biochemical and cellular potencies of the drug. For CT-L activity, proteasome inhibition was dose dependent, with both the initial effect (on Cycle 1, Day 1 - C1D1) and the peak effect of proteasome inhibition increasing in a dose-dependent manner. CT-L inhibition was modest at the lowest dose levels (≤ 0.15 mg/m2), reaching moderate levels after repeat dosing. At intermediate dose levels of 0.3 to 0.55 mg/m2, CT-L inhibition was 31% to 75% on C1D1, rising to 70% to 93% after repeat dosing. At doses of 0.7 mg/m2 and above, inhibition of CT-L activity was usually complete within the first cycle. By contrast, C-L and T-L activities were unchanged or increased in the first cycle, suggesting compensatory hyperactivation in response to effective blockade of CT-L activity. Importantly, however, this response was overcome by repeat dosing with MRZ, and inhibition of T-L and C-L activity was noted across all dose levels. For C-L activity, treatment with MRZ at the recommended Phase 2 dose (RP2D) of 0.5 mg/m2 with twice-weekly dosing resulted in inhibition of up to 39% by Cycle 2 and was maintained, when tested, through Cycles 4 and 6. Treatment at the RP2D of 0.7 mg/m2 for once-weekly dosing resulted in C-L inhibition of 14% to 37% by the end of the first cycle, rising to 31% to 50% by the end of Cycle 2. Blockade of T-L activity was more robust after multiple cycles of MRZ therapy. Although inhibition of the T-L subunit was absent on C1D1 in patients receiving 0.5 to 0.55 mg/m2, inhibition of up to 80% was achieved by Cycle 2 and maintained for the duration of treatment. At the once-weekly RP2D of 0.7 mg/m2, T-L inhibition of 29% to 56% was achieved by the end of the first cycle, rising to 64% to 78% by the end of Cycle 2.Conclusions: The PD activity of MRZ against all three proteolytic subunits was assessed in patients with MM, solid tumors, and advanced lymphomas. At the twice-weekly and once-weekly RP2Ds, complete inhibition of CT-L activity was observed within 1-2 cycles of therapy, but accompanied by compensatory hyperactivation of the C-L and T-L subunits. This phenomenon in red cells suggests that it may be due to allosteric interactions within the catalytic core of the 20S proteasome rather than de novo synthesis of additional proteasomes. Ongoing MRZ therapy was able to overcome this adaptive response, resulting in robust pan-subunit proteasome inhibition within 2-6 cycles, most probably due to the cumulative effect of multiple exposures to MRZ. Due to their reversible binding mode (bortezomib, ixazomib) or monospecificity for the CT-L site (carfilzomib, oprozomib), other clinical PIs are predicted to lack this capability. This unique property of MRZ may explain in part its clinical activity in patients with MM resistant to both bortezomib and carfilzomib. DisclosuresOff Label Use: marizomib treatment for multiple myeloma and solid tumours.. Harrison:AbbVie: Research Funding; Janssen: Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding. Burrows:Triphase Accelerator Corporation: Consultancy. Reich:Triphase Accelerator Corporation: Consultancy. Trikha:Triphase Accelerator Corporation: Employment.

Phase 1 clinical trial of marizomib (MRZ, NPI-0052) in patients with advanced malignancies including multiple myeloma: study NPI-0052-102 (NCT00629473) final results

PO-332 Phase 1 clinical trial of marizomib (MRZ, NPI-0052) in patients with advanced malignancies including multiple myeloma: study NPI-0052-102 (NCT00629473) final results S.J. Harrison, L. Catley, T. Price, M.J. Millward, P. Padrik, C.R. Underhill, P.K. Cannell, F.J. Burrows, S.D. Reich, M. Trikha, A. Spencer The Peter MacCallum Cancer Centre; Mater Adult Hospital; Queen Elizabeth Hospital; Sir Charles; Tartu University Hospital; Border Medical Oncology and University of New South Wales; Royal Perth Hospital; Triphase Accelerator; Alfred Health-Monash University

Synergistic Anti-Myeloma Activity of a Proteasome Inhibitor Marizomib and IMiD® Immunomodulatory Drug Pomalidomide

Background and Rationale: Proteasome inhibitor bortezomib is an effective therapy for the treatment of relapsed and refractory multiple myeloma (RRMM); however, prolonged treatment can be associated with toxicity, peripheral neuropathy and drug resistance. Our earlier studies showed that a novel proteasome inhibitor marizomib is distinct from bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities (Chauhan et al., Cancer Cell 2005, 8:407-419). We also showed that marizomib triggers synergistic anti-MM activity in combination with lenalidomide (Chauhan et al., Blood 2010, 115:834-45). Pomalidomide, like lenalidomide, is an analogue of thalidomide with potent immunomodulatory activity, and has been approved by FDA for treatment of RRMM patients who have received at least two prior therapies including lenalidomide and bortezomib and showed disease progression on or within 60 days of completion of the last therapy. Approval of treatment is based on progression-free survival. Here we utilized in vitro and in vivo models of MM to examine the anti-MM activity of combined marizomib and pomalidomide.Materials and Methods:MM celllines, patient tumor cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of marizomib and pomalidomide. Cell viability, apoptosis, and migration assays were performed using WST/MTT, Annexin V staining, and Transwell Inserts, respectively. Synergistic/additive anti-MM activity was analyzed by isobologram analysisusing “CalcuSyn” software program. Proteasome activity was measured, as previously described (Chauhan et al., Cancer Cell 2005, 8:407-419). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. MM.1S-tumor-bearing mice were treated with vehicle control, marizomib, pomalidomide or marizomib plus pomalidomide at the indicated doses for 21 days on a twice-weekly schedule for marizomib and 4 consecutive days weekly for pomalidomide. Statistical significance was determined using a Student’s t test. Pomalidomide was purchased from Selleck chemicals, USA; and marizomib was obtained from Triphase Inc., USA.Results: MM cell lines (MM.1S, MM.1R, INA-6, RPMI-8226, Dox-40, U266, LR5, ANBL6.WT, and ANBL6.BR) and primary patient MM cells were pretreated with DMSO control or with pomalidomide for 24h; marizomib was then added for an additional 24h, followed by assessment of cell viability. A significant decrease in viability of all cell lines and patient cells was observed in response to treatment with combined low doses of marizomib and pomalidomide, compared with either agent alone. Isobologram analysis confirmed the synergistic anti-MM activity of these agents (CI < 1.0). Tumor cells from 5 of 7 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, the cytotoxicity of combination therapy was observed in MM cell lines sensitive and resistant to conventional (dex, doxorubicin, melphalan) and novel (bortezomib) therapies. No significant decrease in viability of PBMCs from normal healthy donors was observed in response to treatment with combined low doses of marizomib and pomalidomide, suggesting selective anti-MM activity and a favorable therapeutic index for this combination regimen. Furthermore, marizomib plus pomalidomide inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies showed that marizomib plus pomalidomide-induced apoptosis was associated with: 1) activation of caspase-8, caspase-9, caspase-3, and PARP; 2) downregulation of Cereblon, IRF4, c-Myc, and Mcl-1; and 3) enhanced inhibition of chymotrypsin-like, caspase-like and trypsin-like proteasome activities versus single agent alone. Furthermore, combined low doses of marizomib and pomalidomide blocked migration of MM cells and angiogenesis. In vivo studies using a subcutaneous human MM xenograft models show that combined low doses of marizomib and pomalidomide are well tolerated, inhibit tumor growth, and prolong survival.Conclusion: Our preclinical studies in MM disease models support a clinical trial of combined marizomib and pomalidomide to improve outcome in patients with relapsed and refractory MM. DisclosuresRichardson:Oncopeptides AB: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees. Trikha:Triphase Accelerator: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

Anti-Myeloma Activity of a Novel Glutaminase Inhibitor CB-839

Background and Rationale: Cancer cells possess different metabolic requirements than normal cells, such as increased fatty acid synthesis and increased rates of glutamine metabolism, which facilitate their growth and survival. Glutamine is one of the key metabolites required by different cancer cell types for their survival. It is a precursor for α-keto glutarate (αKG) of tricarboxylic acid cycle. Intracellular glutamine is metabolized by a biochemical process glutaminolysis: glutamine is converted to glutamate by glutaminase (GLS), followed by glutamate conversion to αKG by glutamate dehydrogenase (GDH). Three mammalian GLS have been identified: the Liver-type (LGA or GLS2), the Kidney-type (KGA) and Glutaminase C (GAC). Elevated GAC mRNA levels have been detected in gliomas, colorectal carcinomas, adenomas and breast tumor cell lines. GLS activity is linked to tumor growth, since its inhibition by small molecule inhibitors or siRNA knockdown suppresses tumor growth in solid tumors. Elevated glutaminolysis confers drug resistance. These findings suggest that targeting GLS activity inhibits oncogenic transformation and overcomes drug resistance. Here we evaluated anti-MM activity of CB-839, a novel potent orally bioavailable inhibitor of both KGA and GAC splice variants of glutaminase.Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of CB-839. All studies involving human samples were performed under approved protocols at Dana-Farber Cancer Institute. Cell viability was assessed by WST-1 (Chemicon International) assay, as per manufacturer’s instructions. MM cells from patients were isolated by CD138-positive selection using CD138 microbeads and the auto MACS magnetic cell sorter. Total cell lysates were subjected to immunoblot analysis. HUVEC cell angiogenesis assay and transwell cell migration assays were performed, as previously described (Chauhan et al., Blood, 2010, 115(4): 834-45). Glutamine and glutamate determination kit was purchased from Sigma, USA. Drug source: CB-839 was obtained from Calithera Biosciences, USA.Results: KGA and GAC are highly expressed in MM cell lines than in normal PBMCs. We first confirmed the functional specificity of glutamines (GLS) inhibitor CB-839 using MM cells. Specifically, a significant increase in glutamine and a concomitant decrease in glutamate levels were observed in CB-839-treated RPMI-8226 MM cells (p < 0.05; n=3). Moreover, treatment of MM cells with CB-839 significantly decreased both basal and active α-keto glutarate levels (p < 0.05; n=3). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, U266, ARP-1, INA-6, ANBL6.WT, ANBL6.R, and LR5) and primary patient cells for 72h significantly decreased their viability (IC50 range 1.25 μM-10 μM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for CB-839. Conversely, pretreatment of MM cells with α-keto glutarate, a downstream product of glutamine breakdown abrogates anti-MM activity of CB-839. Moreover, CB-839 inhibits proliferation of MM cells even in the presence of BM stromal accessory cells. Mechanistic studies show that CB-839 triggered MM cell death occurs in a caspase-dependent manner, associated with induction of autophagy in MM cells via inhibition of PI3K/Akt/mTOR signaling pathway without concurrent activation of mTORC1. CB-839 induced autophagy was evident by increased levels of autophagy markers Beclin-1, Atg-7 and LC3B. Combining CB-839 with a biochemical inhibitor of autophagy 3-methyl adenine or chloroquine triggered antagonism, suggesting that both autophagy and caspase-mediated apoptotic pathway contribute to anti-MM activity of CB-839. Furthermore, CB-839 blocked migration of MM cells and angiogenesis. Finally, combination of CB-839 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity.Conclusion: Our preclinical studies showing efficacy of CB-839 in MM disease models provide the framework for a clinical trial of CB-839, either alone or in combination, to improve outcome in relapsed and refractory MM patients. DisclosuresMunshi:Ocopep: Consultancy, Equity Ownership, Patents & Royalties; Sanofi-Aventis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Celgene: Consultancy. Richardson:Oncopeptides AB: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

Anti-Myeloma Activity of a Novel Free Radical Inducer Rrx-001

Background and Rationale: Multiple Myeloma (MM) remains incurable despite the advent of novel drugs, highlighting the need for further identification of factors mediating disease progression and resistance. The bone marrow (BM) microenvironment confers growth, survival, and drug resistance in MM cells. Studies to date suggest an important role of BM hypoxia (low oxygenation) in MM cell survival, drug resistance, migration, and metastasis. Therapies targeting the MM cell in its BM milieu under hypoxic conditions may therefore achieve responses in patients resistant to various therapies. Recent studies led to the development of a novel aerospace-industry derived Phase 2 molecule RRx-001 with epigenetic and NO-donating properties. RRx-001 generates reactive oxygen and nitrogen species (RONS), which induces oxidative stress in tumor cells. Importantly, RRx-001 is also a potent vascular disrupting agent, which further provides rationale for utilizing RRx-001 as a therapeutic agent since tumor-associated angiogenesis is a characteristic of MM. A Phase I clinical trial has shown RRx-001 to have antitumor activity in heavily pretreated cancer patients and to be safe and well tolerated with no dose-limiting toxicities (Reid et al. J Clin Oncol 32:5s, 2014 suppl; abstr 2578). Here we examined the anti-MM activity of RRx-001 using in vitro and in vivo models of MM.Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of RRx-001 alone or in combination with other agents. Drug sensitivity, cell viability, apoptosis, and migration assays were performed using WST, MTT, Annexin V staining, and transwell Inserts, respectively. Synergistic/additive anti-MM activity was assessed by isobologram analysisusing “CalcuSyn” software program. Signal transduction pathways were evaluated using immunoblotting. ROS release, nitric oxide generation, and mitochondrial membrane potential was measured as previously described (Chauhan et al., Blood, 2004, 104:2458). In vitro angiogenesis was assessed using matrigel capillary-like tube structure formation assays. DNMT1 activity was measured in protein lysates using EpiQuik DNMT1 assay kit. 5-methyl cytosine levels were analyzed in gDNA samples using methylflash methylated DNA quantification kit from Enzo life sciences; USA. For xenograft mouse model, CB-17 SCID-mice were subcutaneously inoculated with MM.1S cells as previously described (Chauhan et al., Blood, 2010, 115:834). Statistical significance of data was determined using a Student’st test. RRx-001 was obtained from RadioRx Inc., CA, USA; bortezomib, SAHA, and pomalidomide were purchased from Selleck chemicals, USA.Results: Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, OPM2, H929, Dox-40 ARP-1, KMS-11, ANBL6.WT, ANBL6.BR, and LR5) and primary patient cells for 24h significantly decreased their viability (IC50 range 1.25nM to 2.5nM) (p < 0.001; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for RRx-001. Tumor cells from 3 of 5 patients were obtained from patients whose disease was progressing while on bortezomib, dexamethasone, and lenalidomide therapies. Moreover, RRx-001 inhibits proliferation of MM cells even in the presence of BM stromal cells. Mechanistic studies show that RRx-001-triggered apoptosis is associated with 1) induction of DNA damage response signaling via ATM/p53/gH2AX axis; 2) activation of caspases mediating both intrinsic and extrinsic apoptotic pathways; 3) increase in oxidative stress through release of ROS and generation of NO; and 4) decrease in DNA methyltransferase (DNMT1) enzymatic activity and global methylation levels. Furthermore, RRx-001 blocked migration of MM cells and angiogenesis. In vivo studies using subcutaneous human MM xenograft models show that RRx-001 is well tolerated and inhibits tumor growth. Finally, combining RRx-001 with bortezomib, SAHA, or pomalidomide induces synergistic anti-MM activity and overcomes drug resistance.Conclusion: Our preclinical studies showing efficacy of RRx-001 in MM disease models provide the framework for clinical trial of RRx-001, either alone or in combination, to improve outcome in relapsed and refractory MM patients. DisclosuresRichardson:Oncopeptides AB: Membership on an entity’s Board of Directors or advisory committees; Celgene: Membership on an entity’s Board of Directors or advisory committees; Millennium: Membership on an entity’s Board of Directors or advisory committees. Oronsky:RadioRx Inc, : Employment. Scicinski:RadioRx Inc,: Employment. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.