Kumar, Shaji

Abstract Background: Multiple myeloma remains incurable with current approaches and newer therapies are needed to improve the outcome of these patients. While monoclonal antibody based therapies have been successful in some of the hematological malignancies, such approaches have had limited efficacy in the setting of myeloma. Thymoglobulin (polyclonal rabbit antithymocyte globulin, Genzyme) (Thymo) has been extensively evaluated in the setting of allogeneic blood and marrow transplantation and solid organ transplants. Given the polyclonal nature of this product, with antibodies against different B cell antigens, we evaluated the in vitro and in vivo activity of Thymo in myeloma. Methods: MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum supplemented with L-Glutamine, penicillin, and streptomycin. The KAS-6/1 cell line was also supplemented with 1 ng/ml IL-6. Cytotoxicity following drug treatment was measured using the MTT viability assay. Apoptosis was measured by flow cytometry using Annexin V/PI in cell lines and Apo 2.7 in primary patient plasma cells. Shifts in expression of a variety of different B cell and plasma cell antigens were examined on several different myeloma cell lines following Thymo treatment in order to identify the potential antigenic targets. In vivo activity of thymo was evaluated in a SCID plasmacytoma model injected with RPMI myeloma cell lines. Results: rATG was cytotoxic in vitro to several MM cell lines (RPMI 8226, U266, OPM1, OPM2) including the IL-6 dependent cell line Kas6/1 with LC50 of around 1 mg/mL. Additionally, thymo was cytotoxic MM cell lines resistant to conventional agents such as doxorubicin (Dox40), melphalan (LR5) and dexamethasone (MM1R). Thymo induced apoptosis in MM cell lines and in patient derived primary myeloma cells. When tested in combination with other anti-myeloma agents an additive effect was seen with doxorubicin, PS341 and melphalan. Using competitive flow cytometry, we identified CD138, CD38, Cd45, CD126, CD49d (VLA4), as well as CD20 as antigens likely to be targeted by Thymo. Tumor bearing mice injected with Thymo at two different doses (5 mg/kg and 10 mg/kg for five days) had significantly delayed tumor growth compared to non-injected mice, and this translated into a better survival for these mice. Mice receiving 10 mg/kg dose had a slower tumor growth compared to 5 mg/kg dose (Figure). Conclusions: Thymoglobulin has promising in vitro and in vivo activity in the setting of myeloma. These studies will provide the rational for future clinical development of this agent in myeloma alone or in combination with other agents. Based on these results, we are in the process of initiating a clinical trial combining Thymo with Melphalan. Figure Figure

Abstract Background: Renal insufficiency of varying severity including end stage renal disease requiring dialysis is often a presenting manifestation of multiple myeloma (MM). While many patients (pts) improve following initiation of therapy for MM, a considerable number of pts proceed to stem cell transplant with less than optimal renal function. It is not clear whether the outcome of their transplants differs from pts with normal renal function. Methods: The MM transplant database was searched to identify pts with a Creatinine clearance (Crcl) of less than 50 ml/min/m2 (study cohort). These pts were then matched to two pts each with a clearance of ≥ 50 ml/min/m2 (control cohort), based on cytogenetic abnormalities (normal vs. abnormal), circulating plasma cells (yes vs. no), age at transplant, % of plasma cells at transplant, plasma cell labeling index, time to transplant from diagnosis and the disease status at transplant, the factors used in that order of importance. Results: Eighty pts with a Crcl < 50 ml/min were identified and matched to 160 pts with Crcl ≥ 50 ml/min. The pts were matched for the above factors and the two groups were similar in terms of their other baseline features except CRP and B2M, both of which were lower in the control group. Pts with reduced renal function were more likely to receive dose reduced melphalan for conditioning (32.5%) compared to the control group (2%; P < 0.0001). The outcomes from the stem cell transplant were similar in both the groups. 96% of pts obtained an objective response to stem cell transplant in each of the groups with no difference in the time to response; and a complete response was seen in 38.5% and 36.6% of the study and control cohorts respectively (P=NS). The median post transplant progression free survival was 18.2 months and 18.4 mos respectively for the study cohort and the control group (P=0.4). However, the median overall survival from transplant was shorter for the study cohort (30.7 mos vs. 60 mos for the controls; P = 0.007; Figure). The time to platelet and neutrophil engraftment was not different between the groups. In terms of transplant related toxicity, the median days hospitalized was higher in the study group (5 days vs. 9.5 days; P=0.003) as was the incidence of grade 3 and 4 mucositis (8% vs. 23%; P= 0.0008). Six pts (7.5%) had treatment related deaths in the study group compared to 3 pts (2%) among the controls (P=0.03), the majority of the deaths being infection related. Conclusion: Here we demonstrate that pts with MM and reduced renal function derive disease free survival comparable to those with normal renal function, despite the increased use of reduced dose Melphalan for conditioning. The toxicity is clearly increased in the presence of abnormal renal function. Interestingly, the overall survival was inferior among the pts with reduced renal function. One possible explanation for this observation is the decreased availability of novel therapies for salvage among pts with reduced renal function as many of them would not be eligible for clinical trials due to reduced renal function. Figure Figure

Abstract Background: Patients with early stage myeloma are typically observed without therapy until symptomatic disease occurs. We have reported early results of therapy with single-agent thalidomide as initial therapy for early stage multiple myeloma in an attempt to delay progression to symptomatic disease (Leukemia2003;17:775–9). We now report the long-term results of this phase II trial. Methods: Thirty-one patients with smoldering or indolent multiple myeloma were enrolled between April 1999–March 2002. All pts had bone marrow (BM) plasma cells >=10% and measurable disease defined as serum monoclonal (M) protein >=2gm/dL and/or urine M protein >=400mg/24 hours. Two patients were deemed ineligible because they were found to have received prior therapy for myeloma and were excluded from analysis. Thalidomide was initiated at a dose of 200 mg/day and increased as tolerated by 200 mg/day every 2 weeks to a maximum dose of 800 mg/day. The dose of thalidomide was decreased as needed to minimize toxicity. Treatment was continued until disease progression or serious toxicity, except for patients achieving less than a minor response (<25% paraprotein reduction) to therapy in whom therapy was stopped at one year. Response was defined as a decrease in serum and urine monoclonal (M) protein by 50% or higher. If the serum M protein was unmeasurable, a 90% or higher decrease in urine M protein was required. Responses need to be confirmed at least 4 weeks apart. Results: Ten patients (34%) had indolent myeloma, including 4 patients with lytic bone lesions, 3 patients with anemia (Hb <11gm/dL), and 3 patients with both anemia and lytic bone lesions. Median follow up of living patients is 5.6 years (range, 4.0–7.1 years). Of the twenty-nine eligible patients, ten (34%) had a partial response to therapy with at least 50% or greater reduction in serum and urine monoclonal (M) protein. Three patients are still on study and receiving therapy. The remaining have gone off-study: seven patients (27%)had progressive disease while on therapy; other reasons for ending study treatment included completed therapy per protocol (2 pts), patient refusal (2pts), toxicity (8 pts), alternate treatment (3 pts), other medical problems (1 pt), died on study (1 pt), and other (2 pts). The median time to progression (TTP) was 3.2 years (95% CI 2.1–5.6 yrs). Median progression free survival (PFS) was 3.0 yrs (95% CI 2.0–5.1 yrs). Median overall survival has not been reached; 5 year survival rate by the Kaplan Meier method is 60%. Major grade 3–4 non-hematologic adverse events at any time during therapy were noted in 34% of patients; this includes 14% with grade 4 toxicity. Grade 3–4 toxicities occurring in more than one patient include neuropathy (2 pts) and sinus bradycardia (2 pts). Conclusions: Thalidomide has activity in asymptomatic myeloma. This approach must be further tested in randomized trials to determine if the survival estimates are superior to observation. Given the activity of thalidomide, trials are also needed with lenalidomide.

Abstract Background: Lenalidomide (Revlimid®) in combination with dexamethasone was approved in the US on June 29, 2006 for the treatment of subjects with multiple myeloma who had received at least one prior therapy. On February 28, 2005 based upon a positive interim analysis of two pivotal placebo-controlled Phase III studies, an independent Data Safety Monitoring Board recommended the studies be unblinded and all subjects in both studies be given access to lenalidomide. In April 2005, the FDA in association with myeloma patient advocacy groups requested Celgene establish an expanded access program to make lenalidomide plus dexamethasone available to subjects with relapsed or refractory multiple myeloma while the treatment was awaiting approval. Aim: To provide lenalidomide to multiple myeloma subjects with a high likelihood of benefit and to obtain additional safety data. Methods: Subjects with relapsed or refractory multiple myeloma that received at least 1 prior therapy were eligible. Subjects received 25 mg lenalidomide plus high-dose dexamethasone in 4-week cycles until disease progression was documented, study drug was discontinued, or lenalidomide became commercially available for this indication. Results: Between September 8, 2005 and July 25, 2006, approximately 1400 subjects in the US and Canada were enrolled into the study. A data snapshot taken March 17, 2006 demonstrated that 746 subjects had been enrolled, median age was 63 years, 60% were male, and 66.5% had Stage III disease. Median time on study was 7.1 weeks (0.1–24.4) and median daily dose was 20.5 mg. At least one Grade 3 or 4 adverse event was reported in 261 (35%) of the 746 subjects. Most commonly reported Grade 3–4 events were neutropenia (7.9% of subjects), thrombocytopenia (6.0%), fatigue (3.6%), anemia (3.5%), pneumonia (3.1%) and hyperglycemia (2.0%). These most commonly reported Grade 3–4 adverse events were the same as those found in the previous pivotal studies, however, their frequencies of occurrence were lower in the current study probably due to ongoing data collection and differences in study maturity. Likewise, the most commonly reported adverse events (all grades) were the same as those reported in the two previous pivotal studies. Conclusion: Preliminary data from this expanded access program in over 1400 subjects with multiple myeloma are consistent with results from two earlier Phase III pivotal studies. The EAP of lenalidomide plus dexamethasone in multiple myeloma represents a model of how government, advocacy groups, healthcare providers and industry can work together to quickly provide treatment to subjects in need while a clearly active treatment regimen is awaiting approval.

Abstract Plasma cell (PC) proliferative disorders vary in behavior ranging from MGUS, a usually indolent disorder, to multiple myeloma, an aggressive disease with poor long-term survival. Detecting PC immunoglobulin (Ig) light chain restriction is commonly used to demonstrate PC clonality and establish the diagnosis. Furthermore, greater degrees of peripheral blood (PB) and/or bone marrow (BM) involvement are associated with more aggressive disease types and, therefore, are adverse prognostic indicators. Flow cytometric immunophenotyping (FCIP) is often used for detecting PC Ig light chain restriction. Shortcomings of FCIP as traditionally performed include its relative insensitivity and its underestimation of PC number. For these reasons, the FCIP PC clonality assessment in our laboratory is currently supplemented with a slide-based immunofluorescence (IF) technique. Recent advances in flow cytometry have led to the development of powerful instruments and reagents, potentially enhancing the ability of FCIP to analyze PC clones. In this study a newly available single tube, 6-color FCIP approach for detecting and enumerating monotypic PCs was compared to the current combined FCIP/IF assay. 94 PB and 124 BM samples were analyzed by combined FCIP/IF and single tube 6-color FCIP assays. For PB, FCIP/IF used 2-color CD38-PE/ CD45-PerCP FCIP to enumerate PCs and slide based IF to detect Ig light chain restriction. For BM FCIP/IF used two 4-color FCIP tubes (Tube1: cytoplasmic (cy)-kappa Ig-FITC/CD20-PE/CD45-PerCP/CD38-APC; Tube 2: cy-lambda Ig-FITC/CD56-PE/CD45-PerCP/CD38 APC) and slide-based IF to confirm clonality. These FCIP assays were performed on a FACSCaliber. The single tube, 6-color FCIP assay used the following fluorochrome-antibody combination: cy-kappa Ig-FITC/cy-lambda Ig-PE/CD138-PerCP-Cy5.5/CD38-APC/CD19-PE-Cy7/CD45-APC-Cy7 with data acquired on a FACSCanto. The results are summarized in Tables 1 & 2, the concordance between the methods was high. All false-negative 6-color FCIP results were attributable to the use of isotonic lysis for cell isolation. When limiting the comparison to the ficolled cells alsoused for FCIP/IF, 6-color FCIP was consistently more sensitive. The 6-color assay also allowed for the discriminition between CD19-positive polytypic PCs and CD19-negative monotypic PCs and the detection of CD45-positive monotypic PC subsets which were often present and which were not identified in the combined FCIP/IF approach. Estimates of PB disease burden generated from the 6-color technique were similar to those determined from the FCIP/IF data which have previously been shown to be of prognositic significance. 6-color FCIP allows for the rapid, sensitive detection of monotypic PCs. The ability of 6-color FCIP to detect phenotypically distinct clonal PC subsets enhances the sensitivity and may provide prognositically significant data through both more complete cell enumeration and charactarization. Given these advantages, 6-color FCIP may eventually supplant traditional FCIP methods of PC analysis. Method Comparsion in BM Bone Marrow 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF Positive 81(65%) 3(2%) 84 FCIP/IF Negative 5(4%) 35(28%) 40 Total 86 38 124 Method Comparsion in PB Peripheral Blood 6-color FCIP 6-color FCIP Positive negative total Positive = clone detected; negative = no clone detected. FCIP/IF positive 24(26%) 7(7%) 31 FCIP/IF negative 9(10%) 54(57%) 63 total 33 61 94

Abstract Abstract 2943 Background: Inhibitor of apoptosis (IAP) proteins represents a conserved group of proteins that are important regulators of cell survival and apoptosis. X-linked IAP (XIAP) is the best studied IAP that inhibits pro-apoptotic caspases 3, 7 and 9. Multiple myeloma (MM) cell lines express high levels of XIAP. The levels of XIAP are further increased when stimulated by cytokines IL6 and IGF-1, both secreted in copious amounts in myeloma microenvironment. The other two main IAP proteins, namely cIAP1 and cIAP2 are not direct inhibitors of caspases. Instead, they modulate the levels of various signaling pathways by ubiquitinating proteins within the pathways. The NFKB pathway could be activated or inhibited by cIAP1 and 2. In MM, deletions of cIAP1 and cIAP2 have been shown to activate non-canonical NFKB pathway, which indicates a possible tumor suppressor role of these proteins. We wanted to investigate the role of the three IAPs by using a small molecule inhibitor. Our studies clearly indicate the importance of inhibiting all the three IAPs for the induction of apoptosis in MM cells. Methods: LCL161 was synthesized by Novartis Inc. (Basel, Switzerland). 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 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 drug in order to study the cell signaling pathways. siRNA to cIAP2 was purchased from Invitrogen and was electroporated into MM1S cells. Results: We first examined baseline levels of cIAP1, cIAP2 and XIAP in several MM cell lines and a few patient cells. We observed that the IAPs were constitutively expressed in MM cells. We then wanted to examine the functional significance of these IAP proteins in MM cells. For this, we used an IAP inhibitor LCL161. We observed that LCL161 was able to induce cytotoxicity and inhibit proliferation of MM cells, albeit with differences observed between cell lines. We then examined the factors contributing to resistance in the less sensitive cell lines. For this we chose H929, a sensitive cell line and MM1S, a less sensitive cell line to LCL161. Upon treatment with LCL161, cIAP1 and XIAP were down regulated accompanied by increase in levels of activated caspases 9, 8 and 3 in both H929 and MM1S cells. Using LCL161 in combination with a caspase 9 or a caspase 8 or a pan caspase inhibitor showed clearly that the extrinsic pathway is more involved in the LCL161 induced cell death process. LCL161, however, was unable to inhibit cIAP2 in the less sensitive cell line MM1S whereas cIAP2 was not found to be expressed in H929 cells. It has been shown that cIAP1 is required for ubiquitination and degradation of cIAP2. Therefore, cIAP1 down regulation by LCL161 could actually be contributing to the lack of down regulation of cIAP2 and the observed resistance to LCL161. In order to test this, we used a siRNA to cIAP2 and transfected it into MM1S cells by electroporation. We observed that the siRNA reduced cIAP2 levels and in combination with LCL161 led to marked increase in cells undergoing apoptosis. We also examined signaling pathways after treatment with LCL161 and observed upregulation of both canonical and non-canonical NFKB pathways and Jak/Stat pathway in MM1S cells and not in H929 cells. Combining LCL161 with a Jak2 specific inhibitor SD-1029 synergized in inducing cell death in MM1S and other cell lines less sensitive to LCL161. We are currently testing this combination in MM patient cells. Conclusion: These studies demonstrate the importance of inhibiting cIAP1, cIAP2 and XIAP together in MM cells. Furthermore, by this study we were able to identify resistance mechanisms that are upregulated due to inhibiting the IAP proteins and the importance of using agents that inhibit the IAPs along with inhibitors of these pathways in inducing apoptosis in MM cells. The findings from these studies form the basis of evaluation of IAP inhibitors in combination with a Jak/Stat pathway inhibitor in patients with MM. 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: Multiple myeloma cells are heterogenous in terms of the expression of CD45 and previous studies have suggested important clinical and biological implications of the CD45 expression. It has been shown that expression of CD45 on myeloma cells has prognostic value and that the CD45 positive cells contain the proliferative fraction of the myeloma cells. We have also demonstrated that the CD45+ population has a higher apoptotic rate compared to CD45− cells. Given the hypothesis that CD45+ cells represent myeloma cells earlier in their development and are more proliferative and more dependent on the marrow microenvironment we studied differences between the two populations in patients with different stages of monoclonal gammopathies and in normal plasma cells. Methods: Fresh bone marrow aspirates were subjected to ACK lysis for removal of RBCs subjected to multicolor flow cytometry to study the expression of different surface antigens on the plasma cells. Plasma cells were identified based on their characteristic CD38/45 expression. The surface expression of CD19 (B cell marker), CD56 (NCAM), CD87, (involved in cellular adhesion, cell motility and angiogenesis), CD126 (IL-6 receptor alpha chain), sVEGF (surface VEGF; surrogate measure of VEGF receptor expression), and CD71 (proliferation marker) were studied and compared between the CD45 + and CD45− cells by gating separately on these populations. Results: Patients with active myeloma (76), smoldering myeloma (32), MGUS/amyloid (44), and normal patients (23) were studied. Not all markers were performed on all patients. Among the patient samples, the expression of CD19, CD87, CD71, CD126, and sVEGF were higher among the CD45+ cells compared to CD45− cells (Table: % of cells with expression). There was no significant difference between the two in terms of the expression of CD56. Among the normal plasma cells, the expression of CD19, CD87, CD126 and sVEGF was greater on the CD45+ cells compared to CD45− plasma cells and CD56 was not different. When the patient cells were compared to normal cells, the expression of CD19 was higher among the normal CD45+ plasma cells and CD56, CD87 and CD126 were less. The expression sVEGF was comparable Conclusion: In this study we demonstrate differences between CD45+ and CD45− plasma cells in patients with monoclonal gammopathies. These findings are important for understanding the disease biology and provide an insight into the functional differences seen between these two populations. The increased expression of CD19 in the CD45+ cells supports the notion that these cells are likely to be at an earlier developmental stage. The comparable CD56 expression is not in accordance with previous studies and needs to be examined in a larger group of patients. Increased expression of sVEGF and CD126 as well as the adhesion molecules is in accordance with increased responsiveness and dependence of the CD45+ cells to VEGF and IL-6 and the increased proliferative rates. These findings indirectly support the hypothesis that CD45 negative cells are less dependent on the marrow microenvironment and the cytokine milieu that it provides.

Abstract Background: Treatment of multiple myeloma has evolved considerably in the past few years with availability of several news drugs as well as increasing use of multidrug combinations. These changes have no doubt led to the improved survival seen among patients with MM. We have previously shown that outcomes of patients intolerant or refractory to one of the IMiDs and bortezomib had a poor outcome. Since that time, other drugs of the same class as well as new classes of drugs have been introduced for the treatment of MM. We designed this retrospective study to estimate the outcomes in patients with relapsed myeloma, who have become refractory to the current generation IMiDs and proteasome inhibitors. Patients and Methods: Patients with relapsed multiple myeloma who have received at least 3 prior lines of therapy, is refractory to both an IMiD (lenalidomide or pomalidomide) AND a proteasome inhibitor (bortezomib or carfilzomib), and has been exposed to an alkylating agent were identified from multiple centers. The time patients met the above criteria was defined as T0, and details of all treatment regimens before and after T0 were collected using electronic CRFs. The study was approved by the IRB at the respective centers. Results: 543 patients were enrolled in this study; median age was 62 years (31-87) and 61% were males. Patients were enrolled from centers in North America (n=181), Europe (n=318), and Asia Pacific (n=44). Patients were diagnosed between 2006 and 2014, the median duration between diagnosis of myeloma and study entry (T0) was 3.1 years (0.3 to 9). The median (95% CI) estimated follow up from diagnosis and from T0 were 61 (57, 66) months and 13 (11, 15) months respectively. The median number of lines of therapy prior to T0 was 4 (3-13), 48% had a prior transplant. The median OS from T0 for the entire cohort was 13 (11, 15) months. For these 462 patients, the median number of recorded regimens was 2 (1-9). The overall response and the depth of response to each line of treatment following T0 are as shown in the table. The median (95% CI) PFS and OS from T0 was 5 (4, 6), and 15.2 (13, 17), respectively. The overall survival for the 81 patients with no treatment post T0 was only 2.1 months. In a multivariate analysis, duration from diagnosis to T0, ISS stage III and number of lines of therapy were all associated with inferior PFS, as well as OS, and in addition, serum creatinine>2 mg/dL at T0 also predicted inferior OS. Conclusions: The study provides the expected outcome following development of myeloma that is refractory to a PI and an IMiD. The outcomes of these patients appear to be better than we had seen historically in patients refractory/ intolerant to bortezomib and IMiDs, highlighting the increased treatment options available for these patients. However, there is decreasing response rate to sequential regimens highlighting the development of drug resistance. The data provides a bench mark for comparison of new therapies that are being evaluated in this disease. Table Table. Disclosures Dimopoulos: Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genesis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Kastritis:Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Terpos:BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Takeda: Consultancy, Honoraria; Genesis: Consultancy, Honoraria, Other: Travel expenses; Amgen: Consultancy, Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria. Hillengass:Sanofi: Research Funding; Novartis: Research Funding; Amgen: Consultancy, Honoraria; BMS: Honoraria; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria. Leleu:TEVA: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; LeoPharma: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Pierre Fabre: Honoraria; Celgene: Honoraria; Novartis: Honoraria; Takeda: Honoraria. Oriol:Janssen: Honoraria, Other: Expert board committee; Amgen: Honoraria, Other: Expert board committee. Cavo:Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Mateos:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Vij:Shire: Consultancy; Takeda: Consultancy, Research Funding; Jazz: Consultancy; Karyopharma: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Bristol-Myers Squibb: Consultancy; Amgen: Consultancy, Research Funding. Lokhorst:Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. van de Donk:Amgen: Research Funding; Janssen: Research Funding; BMS: Research Funding; Celgene: Research Funding. Mark:Onyx: Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ludwig:Amgen: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; BMS: Speakers Bureau; Janssen: Speakers Bureau. Jagannath:Novartis: Consultancy; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy; Celgene: Consultancy; Merck: Consultancy. Usmani:Array: Research Funding; Britsol-Myers Squibb: Consultancy, Research Funding; Skyline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioPharma: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Onyx: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Millenium: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Speakers Bureau. Dytfeld:Janssen Poland: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Moreau:Novartis: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Janssen: Honoraria, Speakers Bureau. Lee:Amgen: Membership on an entity's Board of Directors or advisory committees. Shustik:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. de la Rubia:Celgene: Consultancy; Bristol Myers: Consultancy; Amgen,: Consultancy; Janssen: Consultancy. Durie:Takeda: Consultancy; Amgen: Consultancy; Janssen: Consultancy.

Abstract Background: Triplet regimens combining a proteasome inhibitor, an immunomodulatory drug, and a steroid have been shown to be active and well tolerated in previously untreated MM pts. Extended treatment represents an alternative paradigm to induction followed by ASCT in this setting; however, agents for continuous therapy need to be convenient and well tolerated. The investigational agent ixazomib is the first oral proteasome inhibitor to be studied clinically. Phase 1 studies have indicated single-agent activity in relapsed/refractory MM and shown ixazomib to be well tolerated with a manageable safety profile, including limited peripheral neuropathy (PN) (Richardson PG et al, Blood 2014; Kumar SK, et al, Blood 2014). Weekly ixazomib plus lenalidomide-dexamethasone has been investigated in a phase 1/2 study in previously untreated MM pts (NCT01217957); treatment comprised triplet induction therapy followed by single-agent ixazomib maintenance therapy. Results of induction therapy have been previously reported (Kumar SK, et al, ASH 2012). Here we report phase 2 efficacy and safety data in pts receiving ixazomib maintenance. Methods: In phase 2, pts aged ≥18 yrs with measurable disease and no grade ≥2 PN received oral ixazomib 4.0 mg on d 1, 8, 15, lenalidomide 25 mg on d 1–21, and dexamethasone 40 mg on d 1, 8, 15, 22, for up to twelve 28-d induction cycles, followed by maintenance ixazomib on d 1, 8, 15 every 28 d until progression at the dose last tolerated during induction. Transplant-eligible pts could discontinue from study for ASCT after 6 cycles. The primary objective was CR+VGPR rate. Responses were assessed by IMWG criteria. Adverse events (AEs) were graded by NCI-CTCAE v4.02. Results: Fifty pts were enrolled in phase 2; 29 discontinued during induction (cycles 1–12), primarily to undergo ASCT (n=14) or due to AEs (n=6) or pt withdrawal (n=4). Twenty-one pts received ixazomib maintenance therapy (cycle ≥13); 13 (62%) were male, and median age was 68 (range 34–77) years. At data cut-off (June 11, 2014), pts had received a median of 27 treatment cycles (range 15–32), including both induction and maintenance, with a median treatment duration of 26.6 mos (range 13.4–29.6). Mean ixazomib relative dose intensity was 95% and 89% in the induction and maintenance phases, respectively. The mean ixazomib dose administered during maintenance was 3.6 mg weekly. At data cut-off, 11 (52%) pts remained on ixazomib maintenance. Among 49 evaluable phase 2 pts, 44 (90%) achieved ≥PR, including 29 (59%) ≥VGPR. All 21 pts who received ixazomib maintenance had responded to induction therapy. Overall best confirmed/unconfirmed responses in the 21 pts included ≥CR in 11 (52%, of which 4 [19%] were stringent CRs [sCR]), ≥near-CR in 13 (62%), and ≥VGPR in 15 (71%), plus 6 (29%) PR. Responses were rapid and improved during treatment. Median time to first response (≥PR) was 0.99 mos (range 0.92–5.78) and to best response was 7.46 mos (range 1.02–24.74); 33% of pts improved their response during maintenance, including 2 VGPR to near-CR, 3 VGPR to CR, 1 VGPR to sCR, and 1 CR to sCR. At data cut-off, median duration of response was 26.5 mos (range 5.6–26.6+). Among 15 pts achieving ≥VGPR, 5 (33%) had progressed; median duration of ≥VGPR was 23.0 mos (range 3.7–26.1+). All pts were alive after follow-up of 24.6–30.3 mos, including a median follow-up from start of maintenance of 16.9 mos (range 12.9–18.9). Ixazomib maintenance was well tolerated; 15 (71%) pts had drug-related AEs, with only 2 (10%) pts with grade 3 drug-related AEs (hypokalemia, thrombocytopenia) and no grade 4 AEs. The most common (>1 pt) grade 1–2 drug-related AEs were diarrhea (n=8, 38%), nausea, pain in extremity (each n=3, 14%), anemia, and headache (each n=2, 10%). No PN was reported. Serious AEs were reported in 3 (14%) pts during maintenance, including grade 3 acute myocardial infarction, grade 3 pneumonia, and grade 3 orthostatic hypotension; all were considered not related to treatment. Only 1 pt required an ixazomib dose reduction due to an AE (neuralgia). Conclusions: These data indicate that single-agent ixazomib maintenance for up to 1.5 yrs was feasible and generally well tolerated, improved responses following ixazomib-lenalidomide-dexamethasone induction, and contributed to durable responses (median >2 yrs) in previously untreated MM pts not undergoing ASCT. The results support the ongoing phase 3 trial of ixazomib in the maintenance setting (NCT02181413). Disclosures Kumar: Celgene: Consultancy, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding; Sanofi-Aventis: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Skyline Diagnostics: Membership on an entity's Board of Directors or advisory committees. Off Label Use: Investigational proteasome inhibitor ixazomib in combination with lenalidomide and dexamethasone and as maintenance therapy for patients with previously untreated multiple myeloma.. Niesvizky:Onyx Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Millennium: The Takeda Oncology Company: Consultancy, Honoraria, Research Funding, Speakers Bureau. Lonial:Millennium: The Takeda Oncology Company: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Onyx Pharmaceuticals: Consultancy, Research Funding. Laubach:Novartis: Research Funding; Onyx Pharmaceuticals: Research Funding. Hamadani:Millennium: The Takeda Oncology Company: Research Funding. Stewart:Takeda Pharmaceuticals International Co.: Research Funding; Sanofi: Consultancy; Array BioPharma: Consultancy; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Novartis: Consultancy. Hari:Celgene: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Millennium: The Takeda Oncology Co.: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding. Vescio:Celgene: Speakers Bureau; Millennium: The Takeda Oncology Co.: Speakers Bureau; Onyx Pharmaceuticals: Speakers Bureau. Kaufman:Millennium: The Takeda Oncology Co.: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Onyx: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Spectrum: Consultancy, Honoraria; Merck: Research Funding. Berg:Takeda Pharmaceuticals International Co.: Employment. Liao:Takeda Pharmaceuticals International Co.: Employment. Hui:Takeda Pharmaceuticals International Co.: Employment. Richardson:Millennium: The Takeda Oncology Co.: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 1848 Background: Aurora kinases play an important role in cell division by controlling chromatid segregation. Aberrant functions of aurora kinases result in genetic instability, a condition often seen in cancers. While Aurora A is important in the alignment of the sister chromatids, Aurora B, a spindle fiber associated kinase, heavily influences the equal division of sister chromatids and along with the Aurora C kinase also assists in a regulated cell division. Inhibition of either of the Aurora family kinases affects chromosomal alignment and segregation during the course of cell division resulting in polyploidy, cell growth arrest and ultimately cell death. VEGF has been implicated in the increased angiogenesis in MM patients. Increase in VEGF levels leads to upregulation in the Ras/Mek/Erk pathway and increased angiogenesis, cell proliferation and decreased apoptosis. Inhibiting this pathway has shown to induce apoptosis in MM cells. We therefore investigated the role of a small molecule inhibitor A-1014907 that inhibits both aurora kinases and VEGF stimulated Ras/Mek/Erk pathway. Methods: A-1014907 was synthesized and provided by Abbott Laboratories Ltd. 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, 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 drug in order to study the cell signaling pathways. Results: A-1014907 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested with IC50 values between 50–100nM. Similar extents of inhibition of proliferation was also observed when MM cells were co-cultured with bone marrow stromal cells or HUVEC cells or tumor promoting cytokines IL6, IGF and VEGF. The increase in cytotoxicity was due to apoptotic cell death observed in both MM cell lines and patient cells. Cell cycle assays demonstrated that A-1014907 was able to induce cell cycle arrest at the G2/M stage of cell cycle followed by polyploidy indicative of Aurora B inhibition. We also observed an increase over time the proportion of cells in sub G0/G1 stage indicative of cell death. Western blots were performed to understand the mechanism of action of A-1014907. We observed that A-1014907 was able to significantly down regulate Aurora B activity as measured by pHistone H3 (Ser 10) down regulation. This was accompanied by up regulation of p21 and down regulation of CDK4 and cyclin E both indicative of G2/M arrest. We also observed the ability of A-1014907 to inhibit the Ras/Mek/Erk pathway by measuring levels of pErk post treatment with the drug. A-1014907 potently inhibited pErk levels and this down regulation was also observed when MM cells were co-cultured with either VEGF or HUVEC cells. Furthermore, A-1014907 at sub IC50 doses induced synergistic cell death of MM cell lines when combined with sub IC50 doses of dexamethasone Conclusion and current studies: A-1014907 clearly inhibits the Ras/Mek/Erk pathway and aurora B activity and induces apoptosis in MM cell lines and patient cells. We are currently using siRNA to aurora A in combination with A-1014907. We are also examining the mechanism of action of dexamethasone in combination with A-1014907. All this will help to design clinical trials with A-1014907 either alone or in combination with other anti-MM 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.