Citations

Abstract Introduction: With approval of Daratumumab (Dara), targeting CD38 has become an attractive therapeutic option for various B cell cancers including WM. Using preclinical models of WM we investigated CD38 expression patterns, the impact of drug-resistance and the anti-WM effects of Dara, in vitro. An important aspect of our evaluation was the identification of optimal CD38 expression that correlates with maximal cytotoxic effects of Dara; such an approach has not been attempted previously in WM. Methods: WM cell lines (BCWM.1, MWCL.1 and RPCI.WM1) with isogenic proteasome inhibitor-resistant (CR, resistant to carfilzomib, bortezomib and ixazomib) and ibrutinib-resistant subclones (IR) were used. CD38 expression was measured by flow cytometry and a grading system was developed based on increasing CD38 MFI values with 1+= <50,000, 2+ = >50,000 and 3+ = >100,000. Daratumumab-induced specific lysis due to antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) was measured using the Calcein AM assay at an effector to target ratio (E:T) of 100:1. Peripheral blood mononuclear cells (PBMCs) and complement-containing serum were obtained from healthy human donors. Daratumumab, carfilzomib and ibrutinib were obtained from commercial sources. Results: Expression of CD38 (MFI) was highest in RPCI-WM1 (91295.6) > BCWM.1 (13865.3) > MWCL-1 (9804.19) (Figure 1A.). Intriguingly we observed an interesting change in CD38 expression with induction of drug resistance; all CR clones showed a decrease in MFI (RPCI-WM1/CR; øMFI 55960.5 > MWCL-1/CR; øMFI 3890.0 > BCWM.1/CR; øMFI 1381), while decreased expression was noted in all IR clones except one (MWCL-1/IR; øMFI 6272.96, BCWM.1/IR; øMFI 3076 vs. RPCI-WM1/IR; _MFI 461784.6) (Figure 1B). When we applied our CD38 expression grading system, RPCI-WM1/IR cells classified as Grade 3+, RPCI-WM1 and its CR clones as Grade 2+, while all other WM cells were Grade 1+. We noted that utilization of this grading system was helpful in correlating the extent of cytotoxicity to the level of CD38 expression (Table 1). We next treated WM cells with Dara and observed direct lysis in only RPCI-WM1/CR (43%) > BCWM.1/CR (19%) and RPCI-WM1 (11%) cells. However, when Dara-treated WM cells were cocultured with PBMCs, significantly increased ADCC was noted in RPCI-WM1 (90.3%), RPCI-WM1/IR (88.3%) and RPCI-WM1/CR (68.0%) cells (Figure 1C). Although CDC has been reported as a mechanism of mAb-mediated cell death in WM our evaluation of CDC as a potential effector mechanism in daratumumab-treated WM was negative as expected, highlighting ADCC rather than CDC as the primary cytotoxic mechanism. Conclusion: Our analysis of CD38 in WM preclinical models highlights several important findings: 1.) Expression of CD38 changes as WM tumor clones evolve with induction of drug-resistance; an effect that may also be cell-type specific as evidenced by increased CD38 expression in RPCI-WM1/IR cells, 2.) Daratumumab can exert a direct lethal effect in WM cells but this effect is variable and cell line dependent, 3.) Expression of CD38 is an important factor directing cytotoxicity of Dara however antigen density appears to be an important factor and a grading system can be beneficial for future preclinical and/or clinical studies, 4.) ADCC and not CDC appear to be the prominent mechanism of action and 5.) ADCC-mediated cytolytic activity of Dara is contingent on CD38 expression as evidenced by the most specific lysis being noted in CD38 Grade 2+ and 3+ expressing WM cells (Table 1.). These observations can help direct optimal design of clinical studies with Dara in WM where drug resistance and CD38 expression should be further explored as factors contributing to the clinical efficacy of this new agent. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding.

Abstract Background WM remains an incurable cancer despite recent advances. While a phase II trial has demonstrated efficacy of dexamethasone, rituximab and cyclophosphamide (DRC) in the treatment-naïve (TN) patients, this regimen has not been studied systematically in the relapsed/refractory (R/R) setting. Recent data has suggested the MYD88mutation status in WM patients can serve as a predictive marker with therapies such as ibrutinib. We examined the efficacy and tolerability of up to 6 cycles of DRC, with focus on R/R population and activity of this regimen with respect to the patients' MYD88L265Pstatus. Methods Records of WM patients seen consecutively at Mayo Clinic campuses in Rochester, Arizona and Florida from 01/2007 to 12/2014 were reviewed. MYD88L265P status, as assessed by AS-PCR, was recorded when available. Data obtained from symptomatic patients treated with DRC (dexamethasone 20 mg intravenously followed by rituximab 375 mg/m2 intravenously on day 1 and cyclophosphamide 100 mg/m2 orally bid on days 1 to 5) were analyzed. Time-to-event analyses were performed from DRC therapy using the Kaplan-Meier method. We used the Consensus response criteria (6th International Workshop). Results Of 100 symptomatic patients who received DRC, 50 had R/R WM (40% refractory) and 50 were TN. Table 1 shows the patients' baseline characteristics. In the R/R population, DRC was 2nd line (range 2-8) therapy in 58% of patients. The median IgM levels declined from 3,870 mg/dL to 1,846 mg/dL (p=0.0001) at best response. Median follow-up from DRC was 51 months (95% CI: 38-55). The median time-to-best response was 6.8 (0.5-28) months. Overall response rate (ORR) was 87% [VGPR 4%, PR 64%, MR 19%]. Four patients (9%) achieved SD and 2 patients (4%) had PD. Of the sixteen deaths (32%) noted at time of analysis, 11 were related to progressive disease. The median disease-specific survival (DSS) from DRC was not reached (NR) (95% CI: NR-NR). The median progression-free survival (PFS) and time-to-next-therapy (TTNT) were 32 months (95% CI: 15-51) and 50 months (95% CI: 35-60), respectively. In contrast, in the TN patients the median IgM levels declined from 4,130 mg/dL to 1,250 mg/dL (p=0.0001) at best response; median time to best response was 11 (0.6-47) months; median follow-up from DRC was 30 months (95% CI: 21-36). ORR was 96% [VGPR 17% (n=8), PR 70% (n=32), MR 9% (n=4)]. Of the 7 deaths (14%) at time of analysis, 4 were related to WM progression. Median DSS from DRC was NR (95% CI: NR-NR); median PFS and TTNT were 34 months (95% CI: 23-NR) and NR (95% CI: 37-NR), respectively. Among 29 genotyped patients in whom MYD88 mutation status was available, MYD88L265P was present in 25 patients (8 of 10 TN and 17 of 19 R/R patients). The response rates and the time-to-event outcomes were similar in the MYD88L265P and wild-type patients. Grade ≥3 adverse effects included neutropenia (20%), thrombocytopenia (7%) and infections (3%). Conclusions Similar to the frontline setting, DRC is an active and well-tolerated salvage regimen. In contrast to ibrutinib, DRC offers a less expensive, limited-duration treatment option, with preliminary data suggesting activity independent of patients' MYD88 mutation status. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Kumar:Glycomimetics: Consultancy; Sanofi: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Kesios: Consultancy; Array BioPharma: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy. Dispenzieri:Jannsen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; pfizer: Research Funding; Celgene: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Kapoor:Amgen: Research Funding; Celgene: Research Funding; Takeda: Research Funding.

Abstract Background: Aberrant B-cell receptor (BCR) and MYD88 signaling cooperatively drive Waldenstrom macroglobulinemia (WM) tumor cell survival. To sustain BCR-driven growth and maintain optimal cellular homeostasis, WM cells upregulate proteasome function. We have recently described the role of two distinct 19S proteasome-associated deubiquitinating enzymes (DUBs), USP14 and UCHL5, in maintaining WM-cell viability. Pharmacologic inhibition with b-AP15 (commercial compound) results in WM-cell death and accumulation of high molecular weight ubiquitinated protein conjugates; an event that is phenocopied by siRNA knockdown of USP14 and UCHL5. VLX1570 is a lead-optimized clinical grade analog of b-AP15 with enhanced solubility, stability and target residence time. We investigated the effect of VLX1570 on BCR components, WM cell survival proteins and its in vivo activity in a human xenograft model of WM. Materials: VLX1570 was obtained from Vivolux AB and bortezomib from Sellekchem. Human WM cell lines (BCWM.1, MWCL-1 and RPCI-WM1) and their drug-resistant derivatives were used (total n=12). Bone marrow aspirates from WM-patients and from healthy donors were obtained from the Mayo Clinic biospecimens core. PBMCs from healthy donors were used as comparators where needed. NOD/SCID mice were obtained from Taconic Labs and used for profiling the in vivo properties of VLX1570. Results: We first examined for USP14/UCHL5 protein expression by immunohistochemical analysis in primary tumor tissue from WM patients and compared it to healthy donor cells. WM patient-derived cells exhibited greater staining intensity of both DUBs. Preferential binding of VLX1570 to USP14 and UCHL5 in WM cells was probed in a WM cell using an enzyme-based competition assay (HA-Ub-Vs), which demonstrated time-dependent decrease in labeled USP14 and UCHL5 after treatment with VLX1570. In drug-resistant (bortezomib or ibrutinib-resistant) WM cells, VLX1570 induced >50% tumor-specific apoptosis and displayed synergistic activity with ibrutinib (median CI: 0.5). In addition to ER stress markers, we had previously observed that gene expression of BCR-signalosome components were reduced in VLX1570-treated WM cells (NanoString platform) and this was substantiated at the protein level where p-SYK, SYK and BTK decreased in a time-dependent manner. BCR end-effectors were probed in response to VLX1570 and a decrease in nuclear translocation of NFkB (p65) was noted. USP14 has been shown to control the availability of the chemokine receptor CXCR4, which regulates homing of WM cells to bone marrow. After VLX1570 treatment, we noted a significant decrease in CXCR4 surface receptor expression in all WM cell lines tested and primary WM cells. Lastly, to determine the in vivo anti-WM effects of VLX1570, we injected NOD/SCID mice (xenotransplanted with RPCI-WM1-Luc cells) with VLX1570 (I.P. route, 4.4mg/kg every other day) and measured tumor burden along with pharmacodynamic indicators. VLX1570-treated mice (n=7) demonstrated significantly lower tumor volumes by caliper measurement and bioluminescent signal (p=0.01 - 0.003) as well as reduced serum human-IgM, compared to vehicle-treated mice (n=7). Importantly, mice treated with VLX1570 survived longer vs. vehicle-treated mice (median survival, 62 vs. 44 days, respectively, p=0.0008). No unforeseen toxicities were noted and weight of the mice in vehicle vs. VLX1570-treated mice did not significantly change. IHC analysis in mice tumor tissues demonstrated reduced CXCR4, human-IgM, Ki67 and increased cleaved caspase-3 and ubiquitinated (k48-linked) protein level, the latter serving as a more specific indicator of USP14/UCHL5 inhibition. Conclusions: We provide substantial in vitro and in vivo evidence on the anti-WM effects of a novel first-in-class clinical grade USP14/UCHL5 inhibitor, VLX1570. In addition to anticipated induction of ER stress machinery, we describe for the first time VLX1570-mediated modulation of BCR components and WM cell survival proteins as well as demonstrate its robust activity in an aggressive human xenograft model of WM. These data provide the rationale for a clinical trial of single agent VLX1570 in patients with symptomatic WM. Disclosures Martin: Janssen: Consultancy, Honoraria; Acerta: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Bayer: Consultancy. Gullbo:Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

Abstract Background Waldenstrom macroglobulinemia (WM) is an IgM-associated lymphoplasmacytic lymphoma, first described over seven decades ago. Although WM is typically a disease of the elderly, with a median age at diagnosis of ~ 67 years, approximately 10% of patients are ≤50 years (y) of age at diagnosis. Data for young patients are sparse and the few available studies demonstrate inconsistent findings, with potential overestimation of survival owing to inclusion of patients with smoldering WM. In this case-control study, we evaluate outcomes, prognostic features and impact of changing therapies in a large cohort of young symptomatic WM patients compared to matched older patients seen over the course of the past five decades. Methods The medical records of all WM patients seen consecutively at Mayo Clinic from 01/1960 to 10/2013 were reviewed. Of 1181 patients, 140 (11.8%) were ≤ 50 y of age at diagnosis. A cohort of patients 65 y or older at diagnosis, matched 1:1 by the time of diagnosis, served as the control population. The patients were divided into 3 groups based on the timing of initiation of therapy: Group 1 (1960-1977), Group 2 (1978-1995) and Group 3 (1996-2013). The baseline characteristics were compared. Initiation of frontline therapy was used for all time-to-event analysis using the Kaplan Meier method. Results Younger patients were more likely to present with adenopathy and splenomegaly, have higher IgM levels and hyperviscosity symptoms (Table 1). The median follow-up from the frontline therapy was similar (10.7 y vs. 10 y for the control population). At the time of analysis, 91% of the deaths for the younger cases were WM-related compared to 58% in the control arm (p=0.0001). Younger patients had a better OS with a median disease-specific survival (DSS) of 15.6 y (95% CI: 13-21; 10-y OS of 77%) vs. 11 y (95% CI: 7.8-12; 10-y OS of 51%) for the older patient (p=0.0003). Among the young patients, there was no difference in the median DSS across the 3 groups (p=0.42). However, the median DSS for the control group incrementally improved (p=0.02) over the 3 time periods (Table 1). In the younger patients, no improvement in DSS was noted with the use of either frontline rituximab-based therapy compared to non-rituximab based regimens [median NR (95% CI: 7.6-NR) vs. 15.8 y (95% CI: 13-22) with other regimens, p=0.30], or frontline chlorambucil-based compared to non-chlorambucil based regimens [median 16 y (95% CI: 12-22) vs 15.6 y (95% CI: 12-NR) with other regimens, p=0.73]. In the control group, however, there was significant difference in DSS among patients who received frontline rituximab-based compared to non-rituximab based regimens [median NR (95% CI: 8.3-NR) vs 9.1 y (95% CI: 5.6-12) with other regimens, p=0.04], or frontline chlorambucil-based vs non-chlorambucil based regimens [8 y (95% CI: 5-12) vs 12.3 y (95% CI: 11-NR) in other regimens; p=0.001]. Conclusion Striking differences in presentation are evident in young WM patients compared to their older counterparts. The incorporation of rituximab to the previously existing anti-WM regimens and the transition to non-chlorambucil based regimens has resulted in substantial survival gains in the older WM population over the past five decades. However, such improvement in outcome has not yet been observed in the young patients. The majority of younger patients, despite a protracted disease course, succumb to their disease. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Reeder:Novartis: Research Funding; Celgene: Research Funding; BMS: Research Funding; Millennium: Research Funding. Kumar:Kesios: Consultancy; Janssen: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; AbbVie: Research Funding; BMS: Consultancy; Onyx: Consultancy, Research Funding; Glycomimetics: Consultancy; Millennium: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Noxxon Pharma: Consultancy, Research Funding; Array BioPharma: Consultancy, Research Funding; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dispenzieri:Alnylam: Research Funding; Celgene: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; pfizer: Research Funding; Jannsen: Research Funding. Kapoor:Celgene: Research Funding; Takeda: Research Funding; Amgen: Research Funding.

Abstract Background Bendamustine/rituximab (BR) and dexamethasone/rituximab/cyclophosphamide (DRC) combination therapy are acceptable options for the treatment of WM, both in the frontline or salvage setting. Prospective data, however, are only available in treatment-naïve (TN) WM patients, and no direct comparison between BR and DRC has been reported. We compared outcomes of BR and DRC in relapsed/refractory (R/R) and TN WM patients seen at a single institution. Methods Records of WM patients seen consecutively at Mayo Clinic from 01/2007 to 12/2014 were reviewed. The MYD88L265P status, as assessed by AS-PCR, was recorded when available. The data obtained from symptomatic patients treated with DRC or BR were analyzed. The time-to-event analyses were performed from DRC or BR therapy using the Kaplan-Meier method. We used the Consensus criteria (6th International Workshop) for response assessment. Results Of 160 WM patients, 60 patients received BR (73% in R/R setting) and 100 patients received DRC (50% in R/R setting). In the R/R population, BR was the 2nd line (range 2-11) therapy in 47% of patients, while DRC was 2nd line (range 2-8) in 58% of patients. Rituximab monotherapy was the only prior line of therapy in 8 (20%) patients in the BR group and 20 (40%) in the DRC group (p=0.66). Baseline characteristics were similar in patients treated with BR or DRC (Table 1). Six patients received both BR and DRC during their disease course and overlapped between the 2 cohorts. Among the previous untreated patients, the median IgM levels decreased from 3,785 mg/dL to 724 mg/dL (p=0.0001) at best response with BR, while it decreased from 4,130 mg/dL to 1,250 mg/dL (p=0.001) with DRC. The median time to best response was 6.1 (1-25) months in the BR group and 11 (0.5-47) months in the DRC group (p=0.13). For patients treated with BR, the overall response rate (ORR) was 93% [VGPR 29% (n=4), PR 57% (n=8), MR 7% (n=1)]. One patient (7%) had progressive disease. For patients treated with DRC, ORR was 96% [VGPR 17% (n=8), PR 70% (n=32), MR 9% (n=4)]. One patient (4%) achieved SD. Median follow up in the BR and DRC groups were similar (30 months). The 2-year progression-free survival (PFS) was 88% and 61% in the BR and DRC groups, respectively (p=0.08). The 2-year time-to-next therapy (TTNT) was 88% and 76% in the BR and DRC groups, respectively (p=0.35). Median PFS, TTNT and disease specific survival (DSS) were not reached with either regimen. In the R/R setting, the median IgM levels decreased from 3,880 mg/dL to 659 mg/dL (p=0.0001) at best response with BR, and from 3,870 mg/dL to 1,846 mg/dL (p=0.001) with DRC. The median time to best response was similar [7 (1-39) months with BR and 7 (0.5-28) months with DRC (p=0.77)]. For patients treated with BR, ORR was 95% [CR 3% (n=1), VGPR 38% (n=14), PR 41% (n=15), MR 13% (n=5)]. Two patients (5%) had progressive disease. With DRC, the ORR was 87% [VGPR 4% (n=2), PR 64% (n=30), MR 19% (n=9)]. Four patients (9%) achieved SD and 2 patients (4%) had progressive disease. Median follow up from BR was 32 months and 51 months from DRC (p=0.24). The 2-year PFS was 66% and 53% in the BR and DRC groups, respectively (p=0.08). The 2-year TTNT was 75% and 68% in the BR and DRC groups, respectively (p=0.24). The median DSS in the BR group was 69 months (95% CI: 65-69) and NR (95% CI: NR-NR) in the DRC group (p=0.57). The proportion of patients with R/R disease was significantly higher in the BR cohort (Table 1). In a bivariate analysis of the entire cohort for PFS, incorporating the setting (TN vs. RR) and the regimen involved (BR vs. DRC), the latter emerged as a significant factor (hazard ratio 0.52, p=0.019) in favor of BR. Time-to-event outcomes and response rates were similar in the MYD88L265P and MYD88WT patients. Grade ≥ 3 adverse events were neutropenia (12%), infections (7%) and nausea/vomiting (2%) with BR, and neutropenia (20%), thrombocytopenia (7%) and infections (3%) with DRC. Conclusions Although both DRC and BR regimens show activity and comparable toxicities, BR regimen shows a trend for superior PFS in WM patients, both in the TN and R/R setting. No difference was seen in TTNT. MYD88 L265P mutation status does not appear to impact activity of BR or DRC. Randomized controlled trial(s) are needed to confirm our findings. Disclosures Ailawadhi: Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy. Ansell:BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Reeder:Millennium: Research Funding; BMS: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Dispenzieri:Jannsen: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; pfizer: Research Funding; Prothena: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Alnylam: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees. Kumar:Sanofi: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; AbbVie: Research Funding; Array BioPharma: Consultancy, Research Funding; BMS: Consultancy; Kesios: Consultancy; Glycomimetics: Consultancy; Skyline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Noxxon Pharma: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kapoor:Celgene: Research Funding; Takeda: Research Funding; Amgen: Research Funding.

Abstract Background: Mitochondrial activity is critical to maintain a high cellular proliferation especially in drug-resistant clones. Ibrutinib-resistance (IR) in CLL is associated with highly aggressive clinical behavior, carrying a median survival of ~3 months. While these outcomes are not yet reported in WM, resistance to ibrutinib is foreseeable considering no WM patients achieve a complete remission with BTK inhibitors (reported series). The transition to a highly proliferative drive in the IR clone remains a biological dilemma and understanding this will allow rationale development of therapeutic strategies. It is established that increased cell proliferation is highly dependent on the functional capabilities of the mitochondria. Yet the respiratory proficiencies and adaptation of mitochondrial machinery in the IR state remains unknown. Here we interrogated mitochondrial function and its relevance in IR cells using our WM models. Materials: WM cell lines (BCWM.1, non-IR) and its isogenic IR clone (BCWM.1/IR; 20-fold resistant, without BTK or CXCR4WHIM-like mutations) were used. Mitochondrial respirometry was measured by Seahorse XF96 flux analyzer (Fig. 1a). Drug sensitivity and cytotoxicity were determined by Cell-Titer Glo cell viability assay. Whole exome sequencing (WES) was performed on an Illumina HiSeq2000. Results: IR WM cells were noted to be in G1 arrest with altered morphologic features and growth kinetics. Despite growth arrest, IR cells showed increased basal oxygen consumption rate (OCR) (>30% increase) vs. parental (non-IR) cells. Consistent with this we observed enhanced mitochondrial ATP production suggesting increased mitochondrial utilization by the IR clones. We then measured maximal respiratory potential of WM cells with FCCP pre-treatment, which evaluates cellular potential to respond to increased ATP demand. We observed upregulation of mitochondrial spare respiratory capacity in BCWM.1/IR cells vs. parental BCWM.1 cell. In addition, a >50% increase in non-mitochondrial oxygen consumption/respiration was also noted (Fig.1b, c). To identify potential genomic changes that could explain mitochondrial overdrive, WES of the non-IR and IR cells was performed. Interestingly, this analysis did not uncover any mutational alterations in genes directing cellular oxidative phosphorylation (NDUF, COX1, COQ7, SURF1, etc.), glycolysis (PDK1, HK2, PKM2, ALDOA, etc.) or ATP production (ATP5, ND1, UCP1 and others). We then asked if mitochondrial function impacts therapeutic potential of a specific drug (or a class of drug), focusing specifically on proteasome disruption. First, we observed that proteasome inhibition- independent of the site (20S vs. 19S), retained high cytotoxic potential, wherein IR cells showed exquisite sensitivity to proteasome disruption. Interestingly, VLX1570 (targets 19S proteasome) had a 2-fold lower IC50 in IR clones than its non-IR counterpart. Cell respirometry demonstrated that compared to parental cells, VLX1570 (500nM) treatment significantly (p<0.002) and rapidly (2hr) attenuated basal respiration in IR clones at concentrations half of those needed to achieve similar effects in BCWM.1 cells. A concomitant decrease in ATP production as well as spare respiratory capacity was observed at these time points/drug concentrations (significantly more evident than in BCWM.1 cells). While our focus was mitochondrial bioenergetics and its therapeutic relevance in drug-resistant WM, unexpectedly we observed for the first time, evidence that VLX1570 induces changes in mitochondrial bioenergetics as early as 2hrs leading to increased mitochondrial membrane permeability and cell apoptosis in 6-12hrs. Comparable observations were made with bortezomib (targeting 20S). Conclusions: This is the first report identifying the role of mitochondrial hyper-drive in WM cells with resistance to ibrutinib. Of note, enhancedmitochondrial bioenergetics in the IR state increased sensitivity to proteasome inhibition and this was independent of the site of disruption in the proteasome pathway. Although, WES findings alone were not sufficient in explaining mitochondrial overdrive noted in our system, ongoing analysis of various mitochondrial functions (Fig. 1d) at the gene expression, copy number and epigenetic level may elucidate the underlying biomechanics of mitochondrial disposition in IR WM cells. Disclosures Ansell: BMS, Seattle Genetics, Merck, Celldex and Affimed: Research Funding. Linder:Vivolux, Ab: Other: Shareholder. Ailawadhi:Pharmacyclics: Consultancy; Novartis: Consultancy; Amgen Inc: Consultancy; Takeda Oncology: Consultancy.

Abstract Background: Current tumor profiling analytics provide some insight into the various molecular abnormalities and their individual consequences on oncogenic signaling. However, these analyses are limited by their lack of integration where the combined effect of individual mutations, gene copy number variations and chromosomal aberrations are not consolidated to create the global molecular architecture that supports neoplastic growth, particularly in the context of drug resistance. Consequentially, identities of the preferential oncogenic pathway(s) tumor cells employ to oppose the effects of targeted therapies remain cryptic and unactionable. Here we present a simulation-based method, which not only replicates the molecular architecture of ibrutinib-resistant Waldenstroms Macroglobulinemia (WM, for which ibrutinib is the only FDA-approved agent) in silico, but also predicts cell sensitivity towards existing drugs, which we validated experimentally for potential clinical translation. Materials: We used the newly established human WM cell line, RPCI-WM1/IR, as a surrogate model of ibrutinib-refractory WM. Genomic data including whole exome sequencing (WES) and copy number analysis (CNA) was utilized for the creation of an avatar of RPCI-WM1/IR, which through simulation identified the salient and prominently dysregulated cellular pathways. Importantly, illustrating these pathways highlights common convergence points on increased proliferation and viability. These convergence points were then directly and indirectly targeted by simulated testing of a library of FDA approved drugs and those impacting these dysregulated pathways were nominated. Importantly, this simulation avatar approach not only looks for agents acting on the specific gene mutation, but also predicts the convergence points to be attacked. The personalized simulation avatar technology is a comprehensive functional proteomics representation of the WM physiology network. A standardized library of equations models all the biological reactions such as enzymatic reactions, allosteric binding and protein modulation by phosphorylation, de-phosphorylation, ubiquitination, acetylation, prenylation and others. Results: Several genomic aberrations were used to create the RPCI-WM1/IR simulation avatar. Functional activity (based on mutation or copy number alteration) of several ibrutinib targets or transcription factors associated with BTK activity such as FYN, SP1, BMX and FRK were predicted to be lost. Increased expression of CAV1, which also inhibits BTK mediated signaling, was increased. An increase in CSNK2B, which activates PU.1- a transcriptional target of BTK, was also observed. Of note, no CXCR4 mutations, which have been shown to impact ibrutinib response, were observed. Next, the cytotoxic potential of over 150 FDA approved drug (and some in experimental stages) were simulated individually and in combination on the RPCI-WM1/IR avatar. In silico modeling predicted aberrant activity of aurora kinase A (AURKA) and its associated signaling partners, which could be disrupted with the (AURKA) inhibitor, tozasertib. AURKA activation was predicted as upregulated due to alterations in several genes: RASA1 loss and SOS1 increase --> increased ERK --> increased ETS1 --> increased AURKA. High beta-catenin signaling (high CTNNB1 and FZD1/4 and low AXIN1 and GSK3B) was also shown to increase AURKA. The simulation predictions were experimentally validated in vitro where AURKA inhibition with tozasertib significantly inhibited proliferation of RPCI-WM1/IR cells (IC50~14nM) as well as inducing apoptosis (48hr, 20nM treatment) and cell-cycle arrest. Conclusions: Our data demonstrates the potential of in silico modeling in predicting novel drug targets, allowing guidance in 1.) Delineating operational oncogenic circuits in an ibrutinib-resistant state by reanimation of the molecular architecture in silico, 2.) Calculating the impact of individual genomic abnormalities and their collective influence on maintaining tumor survival and 3.) Performing a rapid in-silico drug-sensitivity screen directed by the pathway analyses, which can be validated experimentally using standard assays. This novel approach holds tremendous potential in creating highly personalized therapies for ibrutinib-refractory WM patients based on unique genetic signatures. Disclosures Vali: Cellworks Group, Inc.: Employment, Equity Ownership. Kumar:Cellworks Group, Inc.: Employment. Singh:Cellworks Group, Inc.: Employment. Abbasi:Cellworks Group, Inc.: Employment, Equity Ownership.

Abstract Background: Ixazomib is an experimental, orally bioavailable, proteasome inhibitor that has demonstrated anti-tumor activity in relapsed multiple myeloma (MM). In the dose escalation studies, ixazomib was tolerated up to a dose of 5.5 mg given every week as a single agent, while a dose of 4 mg was utilized in the combination studies with lenalidomide. We undertook this study to examine the efficacy and tolerability of the two doses of ixazomib in combination with dexamethasone in patients with relapsed MM. Patients and methods: This was a randomized phase 2 study of two doses of ixazomib (4mg; Arm A or 5.5 mg; Arm B) given weekly for three weeks with a week off along with weekly dexamethasone (40 mg) in patients with relapsed MM, who are proteasome inhibitor na•ve (including bortezomib) or have received less than 6 cycles of therapy with bortezomib and had a PR or better with no progression at the time of discontinuation. The primary objective was to determine the confirmed overall response rate (>=PR); secondary objectives included progression free and overall survival. A total of 71 patients were accrued from February 2013 to April 2015; one patient was ineligible. Results: Baseline characteristics were similar in the two arms; median age across the study was 70 years (46-84); 53% were male. Median number of prior therapies was 4 (range 2-6); 90% of the patients had prior IMiDs, 70% had prior transplant and 29% had prior bortezomib. At a median follow up of 10 months, 17 (49%) and 19 (54%) of patients had disease progression in arms A and B respectively with 12 (34%) patients in each arm still continuing on treatment. All patients in each arm were evaluable for response; the overall response rates were 31% in arm A (95%CI: 17-49) and 51% (95%CI: 34-69) in Arm B. The depth of response, event free survival and overall survival are outlined in Table 1. Among the patients with no prior bortezomib exposure the response rates were 38% for Arm A and 52% for Arm B. The treatment was well tolerated with 2 patients in each arm discontinuing treatment for adverse events; there were no on study deaths. A grade 3 or higher AE that was at least possibly related to treatment was seen in 21% and 54% in Arms A and B respectively; with 15% and 37% hematologic and 6% and 29% non-hematologic AEs. The most common attributable toxicities encountered included fatigue, thrombocytopenia, diarrhea and nausea with more grade 3 toxicities among Arm B. Peripheral neuropathy, possibly related to ixazomib, was seen in 55% (only grade 1 or 2) in arm A and 43% (2 patients with grade 3) in Arm B. Toxicities led to dose reduction of ixazomib in 17% and 43% of patients in Arm A and B respectively; the median number of cycles administered were 5 (1-24) and 5 (1-22) respectively. Conclusions: Ixazomib in combination with dexamethasone was well tolerated with significant anti-myeloma activity in this group of patients with relapsed MM. Deep responses including stringent CR were observed. The higher dose of ixazomib appears to be associated with a higher response rate but with higher rate of adverse events requiring dose reductions. Table 1. Treatment outcome in all patients Arm B (4 mg) (N=35) Arm C (5.5 mg) (N=35) Response Rate 31% (95%CI: 17-49) 51% (95%CI: 34-69) No. of Responders 11 18 sCR 0 1 CR 1 0 VGPR 7 8 PR 3 9 MR 5 1 Median Overall Survival1 NA NA 6 Months 100% 100% Median Event Free Survival1,2 8.4 mos (95%CI: 4.3-13.2) 8.2 mos (95%CI: 3.8-16.3) %Event Free at 6 Months 60% (95%CI: 45-81) 60% (95%CI: 45-81) Median Duration of Response1 16.7 mos (95%CI: 9.3-22.0) 16.3 mos (95%CI: 7.0-20.1) Median Time to Response 1.1 mos (range: 0.8-3.6) 1.0 mos (range: 0.8-7.5) CI: confidence interval; mo: month; NA: not attained 1Kaplan Meier 2Event-free survival time is defined as the time from registration to the first of disease progression, death due to any cause, or subsequent treatment for multiple myeloma. Disclosures Kumar: Celgene: Consultancy, Research Funding; Sanofi: Consultancy, Research Funding; Skyline: Consultancy, Honoraria; Onyx: Consultancy, Research Funding; Novartis: Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy.

Abstract Background: The clinical impact of ibrutinib is irrefutable and has been hailed as a revolutionary treatment for several cancers, including mantle cell lymphoma (MCL). Despite high response rates noted with ibrutinib therapy in MCL (ORR, 68%), complete responses are low and durability of remission is suboptimal in MCL vs. CLL or Waldenstrom macroglobulinemia (WM) patients (Cheah et al Ann Oncol 2015). This relative lack of efficacy may be due to MCL cells having a higher proliferation rate and being less indolent in nature than CLL or WM cells. Tumor cells with a high proliferation index rely on optimally functioning protein metabolism/homeostasis systems. In MCL, this observation has been fortuitously exploited with the use of bortezomib, a 20S proteasome inhibitor. Indeed the ubiquitin proteasome system is a validated target and consists of several individual components whose function in MCL has yet to be mapped out. We have recently reported that inhibition of the 19S proteasomal deubiquitinating enzymes (DUBs), USP14 and UCHL5; with the small molecule inhibitor b-AP15 is lethal to drug-resistant WM tumor cells. This intriguing observation, prompted us to define the role of USP14/UCHL5 and their inhibition in a diverse panel of B-cell lymphoma cell lines, including MCL models as well as ibrutinib-resistant MCL cells. Materials: b-AP15 (and its clinical grade analog, VLX1570) was obtained from Vivolux AB and bortezomib from Sellekchem. Human NHL (DLBCL, MCL, Burkitt), Hodgkin lymphoma (HL), multiple myeloma (MM) cell lines as well as established drug-resistant subclones were used in drug-sensitivity screening experiments (total n=25). Results: USP14 and UCHL5 mRNA expression was queried in the CCLE database (Broad-Novartis) and showed highest expression in plasma cell and B-lymphoid tumor cell types. This was also observed at the protein level, wherein analysis of The Human Protein Atlas database revealed moderate to high intensity staining for USP14 and UCHL5 localizing to the cytoplasmic/membranous and nuclear compartments in high-grade NHL tissues. Next, we examined B-lymphoid tumor cell sensitivity to pharmacologic inhibition of USP14/UCHL5 with either b-AP15 or VLX1570 (72hr fluorescein diacetate hydrolysis assay or CellTiter Glo). We observed that MCL cells, including those resistant to ibrutinib (Jeko/IR cell line) were highly sensitive to VLX1570 (median IC50 12 - 25nM). In contrast, MM cells were not as sensitive (IC50's ranging between 58 - 90nM). Nine DLBCL cell lines exposed to equivalent time/concentrations of b-AP15 showed a median IC50 of 311nM (range, 70 - 910nM) and 3 HL models exhibited an IC50 of 531nM (range, 420 - 783nM). No difference in sensitivity to VLX1570 was noted between ABC-type vs. GCB-type DLBCL models. These functional results aligned with prior genomic inquiry where USP14 and UCHL5 gene expression was noted to be higher in MCL cell lines compared to DLBCL and non-hematologic cancer models analyzed (FDR=0.0019, p=0.001), suggesting their more central role in maintaining MCL tumor viability. Indeed MCL tumor survival (particularly ibrutinib-resistant cells) appears linked to USP14/UCHL5 function as treatment of Jeko/IR cells with VLX1570 (250nM, 12hrs) caused marked apoptosis (71.8% annexin-V+ staining) and cleavage of PARP-1. Moreover, the combination of ibrutinib and VLX1570 synergistically induced MCL cell death (median CI: 0.3). Mechanistic studies demonstrated that VLX1570 modulates several proteins critical for MCL survival including cyclin-D1, Myc and CXCR4/CD184. Reduction in cyclin-D1 was also noted to coincide with cell cycle arrest of VLX1570-treated MCL cells. Altogether, these findings suggest that VLX1570 modulates critical cellular signaling pathways utilized by MCL tumor cells, particularly those that are involved in resistance to ibrutinib. Conclusions: MCL remains a challenging malignancy to treat with no effective treatments for ibrutinib-relapsed disease. Our data supports the role of the proteasome-associated DUBs, USP14 and UCHL5, as critical factors involved in ibrutinib-resistant MCL tumor survival. VLX1570 is a potentially effective agent for the treatment of ibrutinib-resistant MCL. Disclosures Gullbo: Vivolux AB: Other: Shareholder. Linder:Vivolux AB: Other: Shareholder.

Abstract Background: Dexamethasone (dex) is an integral part of various anti-myeloma regimens both in the frontline and relapsed/refractory (RR) setting. Several clinical trials have shown that dex adds to the efficacy of novel therapeutic agents for multiple myeloma (MM). Yet, a substantial number of patients are intolerant to even the standard of care low-dose dex (40 mg weekly), especially in heavily pre-treated RRMM, possibly due to cumulative steroid toxicities and thus, require dex dose adjustments. There is a concern of compromising the efficacy of anti-MM regimens by adjusting the dex dose lower. We describe the frequency of dex dose adjustment, characteristics of patients who required it and its effect on patient outcomes in a large prospective clinical trial of pomalidomide (pom) and dex in RRMM (MC0789). Methods: We utilized data from the Pom-dex trial (MC0789), which treated RRMM patients in different cohorts of pom (2 vs. 4 mg; 21 vs 28 days cycle) with fixed dose dex (40 mg orally weekly). Patients were divided into 2 subgroups, with any dose adjustment/elimination of dex vs. not. Continuous and categorical variables were compared between the two groups using two-sample t-test/Wilcoxon rank sum test or Chi-squared test, respectively. Logistic regression and Cox regression models were used to find the univariate and stepwise multivariate predictors of response to treatment and overall mortality, respectively. Kaplan-Meier method was used to estimate overall survival (OS) at 1, 3, and 5 years. All statistical tests were two-sided with the alpha level set at 0.05 for statistical significance. Results: Results from the MC0789 clinical trial have been reported previously. Almost half of all treated patients in this trial had dex dose adjusted (n=175/343; 51%). The median number of cycles for the first dose adjustment in this group was 3 (range 1-62), with 46.5% of patients (n=82) having multiple dose adjustments and majority (58.3%, n=102) within the first 3 cycles of treatment. Patients in the dose adjustment group were significantly older (mean 65 vs 60 years; p=0.0001). Males, Whites and patients diagnosed before 2006 or with normal cytogenetics also had a higher incidence of dex dose adjustment, though this was not statistically significant. Patients with dex dose adjustment had a longer duration of treatment (mean 339 days) as compared to the non-adjusted cohort (mean 123.65 days) (95% CI 160.13, 270.58; p <0.0001). Dex dose adjustment was associated with a higher probability of responding in the clinical trial after a univariate (p<0.001) as well as multivariate (p<0.001) analysis. Patients with dex dose adjustment also had a significantly better OS at 1, 3 and 5 years by univariate (p<0.001) and multivariate (p<0.001) analysis. (Figure 1) The only other significant factor was lesser number of prior regimens (3 or less), associated with a higher probability of response (p=0.002) and survival (p<0.001). Conclusion: Our analysis established that in these heavily pre-treated RRMM patients, appropriate dose adjustment of dex did not compromise their response rates; patients remained on treatment significantly longer and had an improved survival. Majority of patients (58.3%) requiring dex dose adjustment had it instituted early during treatment (<3 cycles), suggesting that early disease progression was less likely to determine dex dose continuation or adjustment. Appropriate dex adjustments should be judiciously employed as they may ensure longer treatment duration with novel anti-MM therapeutic agents, conferring superior response and survival in this difficult to treat MM subgroup. This also paves the path to further optimize dex dose in various anti-MM regimens, especially in these patients who are more likely to be intolerant to it. Disclosures Kumar: Skyline, Noxxon: Honoraria; Celgene, Millennium, Onyx, Janssen, Noxxon, Sanofi, BMS, Skyline: Consultancy; Celgene, Millennium, Onyx, Novartis, Janssen, Sanofi: Research Funding.