Citations

Abstract Abstract 2948 Background: Deletions and mutations in the tumor suppressor protein p53 are an uncommon observation in new multiple myeloma (MM) patients and are observed more commonly in patients with advanced disease. p53 deletion has been observed to correlate with poor overall and progression free survival in MM patients. Wild type p53 modulates the expression levels of a broad array of proteins involved in cell cycle progression, apoptosis ultimately leading to cell cycle arrest and apoptosis. p53 is negatively regulated by MDM2. MDM2 binds to and ubiquitinates p53 marking it for proteasomal degradation. In addition, MDM2 is a direct downstream regulator of p53. Targeting the p53-MDM2 interaction by developing agents that bind to the p53 binding motif of MDM2 and reactivating p53 has therefore been an active area of research. Here, we present results from our pre-clinical studies using AT219, a small molecule inhibitor that binds to MDM2 preventing its interaction with p53. Methods: AT219 was obtained from Ascenta Therapeutics. Stock solutions were made using DMSO and working stock solutions were made using RPMI 1640 media containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Akt1/2 kinase inhibitor (Akti) was purchased from Sigma. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Mitocapture and cytochrome-c assays were also performed to confirm the induction of apoptosis in MM cell lines. In order to study the mechanism of action of the drug, immunoblotting studies were performed on lysates made from cell lines incubated with the drug for various time points. Results: AT219 induced potent cytotoxicity in MM cell lines MM1S, MM1R and H929, all three expressing wild type p53 with IC50 values of 2.5–5μM. Similar effects were observed when the above mentioned cell lines were treated with AT219 and the inhibitory effect of proliferation of these cells were examined. When MM1S or H929 cells were cultured with bone marrow stromal cells (BMSCs) derived from MM patients or with one of the three tumor promoting cytokines implicated in MM (IL6, IGF or VEGF) and treated with AT219, the drug was able to inhibit the proliferation of both cell lines to similar extents as observed when cultured independently without BMSCs or the cytokines. The increase in cytotoxicity was found to be due to cells undergoing apoptosis as observed when MM1S or H929 cells were cultured with AT219 and % apoptotic cells were measured as measured by annexin/PI, mitocapture and cytochrome c assays. AT219 was also observed to induce more potent apoptosis in primary cells obtained from new MM patients with wild type p53 than in cells obtained from relapsed MM patients with wild type p53. AT219 clearly upregulated p53 as observed by performing immunoblots after treatment with the drug in MM1S and H929 cells. In addition, MDM2 and p21 were also found to be significantly upregulated and Bax was slightly upregulated post drug treatment. Bcl2, Mcl1 and Xiap levels were down regulated. In MM1S cells AT219 treatment resulted in a slight down regulation of pAkt (Ser 473). However, in H929 cells we observed a transient upregulation of pAkt following AT219 treatment. This prompted us to test AT219 in combination with Akti on MM cell lines. Our results on both MM1S and H929 cells using AT219 in combination with Akti demonstrated synergy. We are currently testing this combination in primary cells drawn from MM patients with both wild type p53 and those with p53 deletions and mutations. Conclusions: Our studies validate the anti-MM activity of AT219 in MM patients with wild type p53. In addition to using AT219 in combination with Akti, we are testing AT219 in combination with existing anti- MM chemotherapeutic agents. Interesting results from our studies will form the basis for clinical evaluation of AT219 as a single agent or in combination with an Akt inhibitor or other agents in MM patients. Disclosures: Kumar: Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.

Abstract Background: Autologous stem cell transplantation is an integral part of myeloma therapy for patients who are able to undergo the treatment. While high dose melphalan has remained the typical conditioning regimen for ASCT in MM, recently there have been efforts to enhance the conditioning therapy by adding newer drugs to melphalan. Addition of bortezomib as well as carfilzomib have been attempted in phase 2 trials. Previous studies have shown that the combination of melphalan and total body irradiation (TBI) is an effective conditioning regimen. Pre-clinical studies have shown that bortezomib can sensitize MM cells to radiation, and based on this finding, we designed a clinical trial to examine the feasibility of combining low dose TBI with a combination of melphalan and bortezomib. Patients and Methods: The primary goal of the phase 1 study was to determine the maximally tolerated dose (MTD) of bortezomib that can be added to high dose melphalan and low dose TBI as part of conditioning chemotherapy in patients undergoing an ASCT for MM. The secondary goals were to examine the toxicities associated with addition of bortezomib to high dose melphalan and TBI in patients with MM and to determine the progression free rate at 1 and 2 years. Patients eligible to receive full dose melphalan conditioning regimen for ASCT were included as long as they had no more than 2 prior regimens and had not received a prior transplant. An accelerated design that was later converted to a standard 3+3 cohort design was used for dose escalation. Three patients were treated at a given dose level combination and observed for at least 36 days to assess toxicity. Melphalan 80 mg/m2 was given days -4, -3, with bortezomib at increasing doses given on day -5, -2 along with TBI of 200 cGY given on the same days of bortezomib, timed after bortezomib administration. Stem cells were infused day 0. Results: Eleven patients were enrolled; 6 were males, and the median age was 60 years. Median time from diagnosis was 5.4 months (range 3.4-7.0). Four dose levels were explored with increasing doses of bortezomib (0.7 mg/m2 to 1.6 mg/m2). No dose limiting toxicities were observed. The dose levels and the best response after ASCT are as shown in the Table. The overall response rate was 100%, including 7/11 with a CR or sCR. With a median follow-up of 12.5 months, two patients have progressed; median OS was not reached. Toxicity was mostly hematologic, with other AEs being grade 3 or less and attributable to the conditioning therapy. All patients engrafted their counts adequately. Conclusion: The combination of melphalan, bortezomib, and low dose TBI is a safe conditioning regimen in patients with MM with good efficacy. The regimen should be further examined in the phase 2 setting. Table Table. Disclosures Kapoor: Celgene: Research Funding; Amgen: Research Funding; Takeda: Research Funding. Kumar:Noxxon: Consultancy, Honoraria; Celgene: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; BMS: Consultancy; AbbVie: Research Funding; Janssen: Research Funding; Skyline: Consultancy, Honoraria.

Abstract Background: Multiple myeloma (MM) cells evade apoptosis through multiple mechanisms thus enabling it to evade therapy. The Bcl2 family of anti-apoptotic proteins is aberrantly expressed in MM cell lines and patient cells. Yet, pharmacological intervention of this family appears to have significant activity only in molecular subgroups of MM patients. This clearly suggests alternate mechanisms of overcoming apoptotic signals in MM cells in addition to the Bcl2 family, through proteins such as IAPs. We have previously shown that simultaneous inhibition of the three major IAP proteins, namely cIAP1, cIAP2 and XIAP is required to induce pronounced apoptosis in MM cells. However, IAP inhibition results in apoptosis in only some MM cell lines and patient cells. Given that levels of Bcl2 family proteins are unaffected by IAP inhibition, we hypothesized that combined inhibition of the IAP proteins using a SMAC mimetic LCL161 and the Bcl2 family proteins using a pan-Bcl2 inhibitor obatoclax (OBX) will lead to more pronounced and synergistic cell death in a broader subgroup of MM patients. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). OBX was purchased from Selleckchem (Houston, USA). Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum (20% serum for primary patient cells) supplemented with L-Glutamine, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drugs in order to study the cell signaling pathways and a Results: LCL161/OBX combination induced synergistic cytotoxicity and anti-proliferative effects on a broad range of human MM cell lines, including drug resistant cell lines like DOX40 and MM1R. Components of the bone marrow microenvironment including bone marrow stromal cells and tumor promoting cytokines (VEGF, IGF and IL6) were unable to protect MM cells from the effects of the drug combination. We saw a time dependent increase in apoptosis, with the combination inducing significantly more apoptosis than either of the single agents alone. Examining the mechanism of action of the drug combination showed clear inhibition of the IAP proteins, activation of caspases 9, 8, 3 and Bid by LCL161 and the combination and up regulation of the pro-apoptotic proteins Bim, Bid, Puma and Noxa and accumulation of LC3-II by OBX and the combination. Using chloroquine along with the OBX, we were able to demonstrate that OBX induced protective autophagy and the addition of LCL161 was able to overcome this protective effect induced after single agent OBX treatment. Since protective autophagy can be induced by the ER stress response, we then examined the expression levels of proteins involved in this pathway. We observed clear induction of ER stress mediated UPR pathway by both the drugs. However, LCL161 and OBX induced different branches of the UPR pathway. OBX activated the ATF6 and pErk/peif2α/ATF4 branches of the UPR, both of which have been implicated in cell survival during ER stress. ATF4 under irrecoverable ER stress can lead to increase in transcription of CHOP and cause apoptosis. We therefore examined levels of CHOP and observed no induction of CHOP post treatment with either of the drugs or the combination. LCL161, however differentially modulated the IRE1 branch of the UPR by down regulating Xbp-1 splicing, which is a pro survival activity of IREI and up regulating pJNK, which indicated a pro-apoptotic activity induced by IRE1 post irrecoverable ER stress This indicated that the ER stress induced apoptosis is triggered by LCL161, which might be important to overcome the ER induced protective effects induced by OBX. Conclusion: Taken together, our studies indicate that LCL161/OBX combination induces synergistic cell death through modulation of apoptosis, authophagy and the ER stress response. Disclosures No relevant conflicts of interest to declare.