ALBERT

Description: The proteasome is a multi-catalytic proteinase complex that is integral to intracellular proteolysis, and plays a key role in many cell functions. Targeting the proteasome with small molecule inhibitors has been validated as a rational therapeutic strategy for patients with relapsed/refractory myeloma with the approval of the first proteasome inhibitor, bortezomib (VELCADE®), for this indication. Additional studies are ongoing to better define the role of this agent in myeloma and other diseases, including non-Hodgkin’s lymphoma. Since bortezomib is a reversible proteasome inhibitor, we considered the possibility that an irreversible agent might have novel, potentially attractive properties. To begin to evaluate this hypothesis, we have studied the efficacy of a novel epoxomicin-related proteasome inhibitor, PR-171, which binds irreversibly and with a high degree of specificity in vitro to the chymotrypsin-like subunit of the proteasome. PR-171 was able to inhibit proliferation of both interleukin (IL)-6-dependent ANBL-6 and KAS-6 cell lines, as well as IL-6-independent models, including RPMI 8226 and U266 cells, in a concentration- and time-dependent fashion. IL-6-dependent cells generally displayed a greater sensitivity to PR-171-mediated effects than IL-6-independent cells. Experiments modeling the in vivo pharmacokinetics of proteasome inhibitors, with a one-hour pulse of drug followed by a washout, showed that PR-171 indeed inhibited the chymotrypsin-like activity of the proteasome without effects on other proteasome proteases. Inhibition of cell proliferation was associated with an induction of programmed cell death, as judged by the appearance of apoptotic oligonucleosome DNA fragments, as well as by the activation of caspase-3. This common effector caspase was activated by both the extrinsic and intrinsic pathways, in that both caspase-8 and caspase-9 were potently induced. Additionally, pulse treatment of PR-171 induced activation of c-Jun-N-terminal kinase, a key signaling molecule in stress-induced and proteasome inhibitor-induced apoptosis. Other members of the stress response-signaling pathway, including heat shock protein-70 and mitogen activated protein kinase phosphatase-1, were induced as well. Finally, both continuous and pulse treatment with PR-171 was also able to inhibit proliferation in freshly purified patient-derived multiple myeloma plasma cells, including isolates from patients with both newly diagnosed, previously untreated disease, as well as isolates from patients who had progressed on other standard therapies, including bortezomib. Importantly, PR-171 was active in both myeloma cell line models and patient-derived samples with chromosome 13 abnormalities. Taken together, these data indicate that PR-171 is a promising, novel proteasome inhibitor with activity against models of multiple myeloma, providing a rational basis for its translation into the clinic.

Description: Introduction: Kinesin spindle protein is a mitotic kinesin that is expressed only in proliferating cells and plays a key role in spindle pole separation, formation of a bipolar mitotic spindle, and thus in satisfaction of the mitotic checkpoint. Ispinesib (SB-715992) is a potent and selective inhibitor of kinesin spindle protein with a Ki of 0.6 nM, has cytotoxic activity at less than 10 nM in a spectrum of tumor cell lines, and disrupts the assembly of functional bipolar mitotic spindles. Methods: This study sought to examine whether spindle disruption by inhibition of kinesin spindle protein with ispinesib may have therapeutic potential in the treatment of multiple myeloma. Results: Ispinesib reduced cell viability in both interleukin-6-independent (RPMI 8226 and U266) and interleukin-6-dependent (ANBL-6 and KAS-6/1) models of multiple myeloma in a time- and concentration-dependent fashion. The average IC50 for ispinesib against these cell lines was 3.0 nM, 1.7 nM, 1.8 nM, and 1.8 nM, respectively. Cell cycle analysis showed that ispinesib induced growth arrest of myeloma cells with 4N DNA content (in M phase) within 24-hours. Two days after treatment at a 1 nM concentration, cells were able to recover from M phase arrest and resume normal cycling but, after exposure to 10 nM, treated cells could not escape M phase arrest, and instead entered apoptosis as determined by an increased sub-G1 population. Ispinesib was able to overcome resistance to melphalan in that the IC50 in melphalan-resistant RPMI 8226/LR5 cells (1.6 ± 0.2 nM) was comparable to that in parental RPMI 8226 controls (3.0 ± 0.9 nM). Similarly, ispinesib was also able to overcome dexamethasone resistance and bortezomib resistance. In regard to the latter, KAS-6/VR5 bortezomib-resistant cells (IC50 of 12.5 nM for bortezomib) retained sensitivity to ispinesib (IC50 1.6 ± 0.2 nM). Combination therapy with ispinesib and bortezomib in these cells resulted in enhanced levels of specific apoptosis (24%) that were greater than the sum of either agents alone (7% for ispinesib and 1% for bortezomib), suggesting synergy. Importantly, ispinesib was also active against freshly isolated CD138+ patient-derived multiple myeloma cells, while relatively sparing CD138− cells. Conclusions: Taken together, these studies demonstrate that kinesin spindle protein inhibition with ispinesib was able to induce growth arrest and apoptosis in myeloma cells, and overcome resistance to both conventional drugs and novel agents such as bortezomib. Moreover, the preferential activity against transformed plasma cells with sparing of normal bone marrow cells provides a strong rationale for translation of this agent into the clinic to combat relapsed/refractory multiple myeloma.

Description: Introduction: The proteasome is a large (~2.5 MDa), ATP-dependent, intracellular protease responsible for degrading ubiquitinated proteins as part of the ubiquitin-proteasome pathway. The immunoproteasome is a unique proteasomal variant with distinct catalytic subunits termed low molecular mass proteins that functions predominately in cells derived from hematopoietic precursors, and differs from the constitutive proteasome found in most other cells. Bortezomib (VELCADE®; Millennium Pharmaceuticals, Inc.) is a first-in-class proteasome inhibitor, which is approved for treatment of multiple myeloma patients who have received at least one prior therapy. While the overall safety profile of bortezomib is manageable and predictable, some toxicities, such as peripheral neuropathy, associated with bortezomib treatment can be dose-limiting. These toxicities may be due to the inhibition of all isoforms of the proteasome. Development of immunoproteasome-specific inhibitors (IPSIs) would allow for targeted therapy against cancers arising from hematologic origins, thereby sparing normal tissues, such as gastrointestinal and neurological tissues. Methods: We have identified several novel IPSIs, most notably IPSI-001, with selective activity against the immunoproteasome, which we therefore sought to characterize. Results: Expression of proteins associated with the immunoproteasome (low molecular mass protein-2; 11S Reg-α) was found primarily in cell lines of hematopoietic origin, while solid tumor cell lines exhibited expression of constitutive proteasome proteins (β5; 19S S6′). IPSI-001 exposure induced preferential inhibition of the chymotrypsin-like activity, the rate-limiting step of proteolysis, in hematologic cell lines over solid tumors. This inhibition was associated with an increase in ubiquitinated substrates, activation of c-Jun N-terminal kinase, and accumulation of Bax. IPSI-001 treatment led to preferential induction of apoptosis as evidenced by DNA fragmentation assays and cleavage of β-actin by caspase-3 into an apoptotic marker, fractin. Furthermore, IPSI-001 had potent chymotrypsin-like inhibitory activity in patient samples of chronic lymphocytic leukemia and acute myeloid leukemia. A dose-dependent decrease in proliferation was observed in additional patient samples of acute myeloid leukemia, chronic lymphocytic leukemia, and non-Hodgkin lymphoma treated with IPSI-001. Also, IPSI-001 exposure induced apoptosis in multiple myeloma and chronic lymphocytic leukemia patient samples. In an effort to increase the efficacy of IPSIs, a series of boronic acid analogs were made of several IPSIs. Conversion into the boronate analogs increased the potency by up to 1000-fold against the chymotrypsin-like activity of the immunoproteasome in vitro and in cellulo. Dose-dependent inhibition of proliferation was observed in ANBL-6, KAS-6/1, and Ramos cell lines, which was associated with induction of apoptosis. Conclusions: Studies are ongoing to characterize the specificity and molecular effects associated with IPSI-boronic acid derivatives exposure in immunoproteasome- and constitutive proteasome-containing cell types.

Description: Signal-regulatory proteins (SIRPs) are transmembrane glycoproteins belonging to the immunoglobulin (Ig) superfamily that are expressed in the immune and central nervous systems. SIRPα binds CD47 and inhibits the function of macrophages, dendritic cells, and granulocytes, whereas SIRPβ1 is an orphan receptor that activates the same cell types. A recently identified third member of the SIRP family, SIRPβ2, is as yet uncharacterized in terms of expression, specificity, and function. Here, we show that SIRPβ2 is expressed on T cells and activated natural killer (NK) cells and, like SIRPα, binds CD47, mediating cell-cell adhesion. Consequently, engagement of SIRPβ2 on T cells by CD47 on antigen-presenting cells results in enhanced antigen-specific T-cell proliferation.

Description: Abstract 306 Introduction: Histone deacetylase inhibitors potentiate the efficacy of anthracyclines and proteasome inhibitors in preclinical models of multiple myeloma (MM). We therefore conducted a phase I clinical trial to evaluate the safety of the histone deacetylase inhibitor, vorinostat, in combination with pegylated liposomal doxorubicin (PLD) and bortezomib for patients with relapsed/refractory MM. Patients and Methods: Patients received bortezomib at 1.3mg/m2 on days 1, 4, 8, and 11; PLD at 30mg/m2 on day 4, and escalating doses of vorinostat (200 to 400mg once daily) on days 4 through 11 of a 3-week cycle. Dose escalation followed a standard “3 + 3” design. Patients remained on therapy until disease progression or unacceptable toxicity. Eligibility criteria included an ANC of ≥1.0×109/L, platelets of ≥100×109/L, a CrCl of ≥30 mL/min., and adequate hepatic and cardiac function. The primary objectives of the study were to establish dose limiting toxicities (DLTs) in cycle 1 and the maximum tolerated dose (MTD) for future phase II testing. Results: Nine patients have enrolled thus far at vorinostat dose levels of 200mg (n=3), 300mg (n=4), and 400mg (n=2). Six patients had relapsed disease, while 3 had relapsed disease that was refractory to their last prior therapy. The median age was 56 (range 44–73), median time from diagnosis was 66 months (range 28 to 117), and median prior number of lines of therapy was 2 (1 to 7). Six of 9 patients received prior bortezomib, 3 of whom were refractory, 7 of 9 had received anthracyclines, 9 of 9 were treated with corticosteroids, 8 of 9 were treated with immunomodulatory agents, and 7 of 9 had undergone autologous stem cell transplantation. One patient was removed from the study at the 300mg vorinostat dose level due to a grade 3 infusion reaction with the first dose of PLD and was not evaluable for DLT or response. Otherwise, there have been no DLTs, serious adverse events, or deaths to date. Common non-hematologic toxicities of all grades have included fatigue (44%), constipation (67%), diarrhea (67%), nausea (56%), vomiting (33%), and peripheral neuropathy (56%), the majority of which were grade 1 and 2 in severity. Grade 3 sensory neuropathy was seen in 2 patients. Common hematologic toxicities of all grades have included neutropenia (44%), lymphopenia (44%), and thrombocytopenia (67%). Grade 3/4 neutropenia, lymphopenia and thrombocytopenia were seen in 2, 3, and 2 patients, respectively. Dose reductions for non-hematologic toxicities have been necessary for 3 patients thus far. Using International Myeloma Working Group criteria, 6 out of 7 evaluable patients have responded to treatment, including 1 complete remission (CR), 1 very good partial remission, and 4 partial remissions (PRs). The only non-responder was assigned to the 200mg vorinostat dose level. PRs were seen in 2 of 3 patients with bortezomib-refractory disease. Conclusions: At the dose levels tested thus far, the addition of vorinostat to the PLD/bortezomib backbone is safe and efficacious in relapsed/refractory MM patients, including those with bortezomib-refractory disease. Cumulative constitutional, gastrointestinal, and neurologic toxicities are common but manageable, and will need to be considered when determining the optimal phase II dose moving forward. Enrollment into the 400mg dose cohort continues. Disclosures: Voorhees: Celgene: Speakers Bureau; Millennium Pharmaceuticals, Inc.: Speakers Bureau. Off Label Use: Vorinostat for the treatment of multiple myeloma. Gasparetto:Millennium Pharmaceuticals: Consultancy, Speakers Bureau. Richards:Cephalon, Inc.: Speakers Bureau. Garcia:Millennium Pharmaceuticals: Speakers Bureau; Celgene: Speakers Bureau. MacLean:Novartis: Speakers Bureau. Foster:Genzyme: Consultancy, Research Funding. Shea:Otsuka: Research Funding; Novartis: Consultancy, Research Funding; Millennium Pharmaceuticals: Research Funding; Genzyme: Consultancy, Research Funding; Genetech: Consultancy. Rizvi:Merck: Employment.

Description: HYDROXYUREA REVERSES DYSFUNCTIONAL UBIQUITIN-PROTEASOMAL SYSTEM IN SICKLE CELL DISEASE AND SUPPRESSES POSTTRANSLATIONAL ALTERATIONS IN HEMOGLOBIN AND CELL MEMBRANES Sirsendu Jana, PhD1, Michael Brad Strader, PhD1, Fantao Meng, PhD1,Michael Heaven, PhD2, Arun Shet, MD3, Swee Lay Thein, MD3, and Abdu I. Alayash, PhD1. 1Laboratory of Biochemistry and Vascular Biology, Center for Biologics Evaluation and Research, Food and Drug Administration (FDA), 2Vulcan Analytical, Birmingham Al, 3Sickle Cell Branch, National Heart, Lung and Blood Institute (NHLBI), National Institutes of Health (NIH). Introduction: Intracellular oxidative stress and oxidative modifications of sickle cell hemoglobin (HbS) play an important role in the pathogenesis of sickle cell disease (SCD). We recently reported transgenic mice studies revealing microparticles (MP) proteome differences between SCD and control mice expressing human HbS and HbA, respectively. Hb-dependent oxidative reactions and consequent posttranslational modifications of Hb βCys93 were central to red cell membrane changes that included modification of band3, and ubiquitination of proteins (Jana S et al., JCI Insights, 2018 3:120451). Ubiquitination is an important post-translational modification required for several biological functions including degradation by the ubiquitin-proteasomal system (UPS) proteolytic pathway. Proteins susceptible to oxidative damage are therefore likely degraded by UPS machinery. When these animals were treated with hydroxyurea (HU) they were able to reduce oxidative stress by controlling Hb oxidation side reactions. As a follow-up study, we have characterized human RBC derived MP proteomes of control, untreated and HU treated SCD patient samples to identify the mechanistic basis of how HU treatment reduces oxidative stress. Methods: We used a variety of biochemical and hematological methods to investigate a group of sickle cell disease (SCD) patients (n= 22) who are either on HU treatment (n=10) or without HU treatment (n=12) and a group of ethnically matched controls (n=4). We also performed an additional proteomic analysis on a subset of these patients, including a separate longitudinal study in which SCD patients (n=2) were followed before and after treatment with HU. Results: Immunoprecipitation experiments on RBCs obtained from untreated SCD patients revealed the presence of extensive ubiquitination contrary to those samples obtained from HU treated patients and controls. High proteasomal activity was found in SCD RBCs suggesting accumulated polyubiquitinated proteins found in these samples were not a byproduct of proteasomal inhibition but rather due to imbalance in the redox state of SS RBCs. In addition to Hb oxidation and oxidative modifications (including βCys93), our results revealed differences in the SCD proteome (from both control and untreated groups) including upregulation of phosphorylation and ubiquitination of proteins that are known to interact directly with band3 and are functionally involved in MP formation. Ubiquitination of Hb βLys145 and βLys96 were more abundant in SS patient's samples as well as phosphorylation of band3 (a prerequisite process for band3 clustering and MPs release). As revealed by the separate longitudinal study, HU treatment uniformly reversed ubiquination and phosphorylation of proteins involved in SCD induced MP formation. Conclusion: These mechanistic analyses of SCD RBC derived MPs suggest a potential involvement of ubiquitination and phosphorylation in SCD pathogenesis and provide additional insight into the therapeutic mechanisms of HU treatment. Disclosures No relevant conflicts of interest to declare.

Description: Polymerization of sickle cell hemoglobin S (HbS) is recognized as a key event in the pathophysiology of sickle cell disease (SCD). Repeated HbS polymerization promotes an altered red blood cell (RBC) membrane, hemolysis, and microparticle (MP) formation, which have been shown to play significant roles in the interaction of RBCs with vascular endothelium and progression of vaso-occlusive events. Circulating RBC-derived MPs are elevated in SCD patients and they release a significant portion of their contents including oxidized HbS and heme to the cells of the vasculature. We have recently reported that free HbS oxidizes faster, remains locked in a highly oxidizing form (ferryl) longer, and loses heme faster than normal HbA (Kassa et al., J Biol Chem 290: 27939, 2015). The contributions of HbS higher oxidation states (ferric and ferryl heme) to MP formation, membrane alterations, and heme loss are poorly defined in SCD. RBC-derived MPs (ranging in size between 100-300 nm in diameter) generated by sheer stress or isolated by ultracentrifugation from the plasma (circulating) of SCD patients (N=6), ethnically matched control subjects (N=5), humanized transgenic sickle mice (Townes-SS, N=4), and control wild-type mice (Townes-AA, N=4) were identified by flow cytometry using CD235a glycophorin antibody and annexin V for externalized phosphatidylserine (PS). Time courses of Hb oxidation, obtained during 30 hour incubations of mouse or human MPs were biphasic. The initial levels of oxidized (ferric) Hb (30 to 45%) were slightly reduced within the first ~10 hours, likely due to the presence of RBC residual reductive enzymes within MPs. This was followed by a second phase in which Hb oxidation (ferric Hb) increased linearly and uncontrollably to 65 to 75% of total Hb. SCD MP's contained highly reactive ferryl Hb intermediates, carbonylated membrane proteins, and phosphorylated band 3 proteins. Quantitative proteomic analysis indicated a higher level of protein oxidation in MPs derived from SCD mice and patients. Five-fold higher levels of irreversibly oxidized βCys93 oxidation were found in untreated versus hydroxyurea-treated SCD patients. Intriguingly, HbS β subunits from SCD MPs were ubiquitinated and MPs isolated from untreated SCD patients had 25-fold higher ubiquitination levels than hydroxyurea-treated SCD patients that were comparable to normal controls. MP ubiquitination levels were correlated with HbS and an overall increase in MP oxidative stress, and inversely correlated with HbF. Compared to respective control MPs, incubation of either mice or human SCD MPs with human endothelial cells (HUVEC) activated apoptotic pathways and impacted cellular bioenergetic parameters by lowering mitochondrial oxygen consumption rates to a greater degree in a manner that was correlated with the redox state of Hb iron within MPs. Human endothelial cells incubated with SCD MPs showed greater intracellular reactive oxygen species production and heme oxygenase-1 induction. In summary, Hb transformation to higher oxidation forms is markedly increased in MPs generated from SCD mice and patients, which when incubated with endothelial cells, lead to mitochondrial dysfunction and apoptotic cell death. These mechanistic analyses of RBC-derived SCD microparticles suggest potential anti-oxidative reducing modalities that may interrupt MP heme-mediated pathophysiology in patients with SCD. Disclosures Belcher: Cydan/Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.

Description: The proteasome has emerged as an important target for cancer therapy with the approval of bortezomib, a first-in-class, reversible proteasome inhibitor, for relapsed/refractory multiple myeloma (MM). However, many patients have disease that does not respond to bortezomib, whereas others develop resistance, suggesting the need for other inhibitors with enhanced activity. We therefore evaluated a novel, irreversible, epoxomicin-related proteasome inhibitor, carfilzomib. In models of MM, this agent potently bound and specifically inhibited the chymotrypsin-like proteasome and immunoproteasome activities, resulting in accumulation of ubiquitinated substrates. Carfilzomib induced a dose- and time-dependent inhibition of proliferation, ultimately leading to apoptosis. Programmed cell death was associated with activation of c-Jun-N-terminal kinase, mitochondrial membrane depolarization, release of cytochrome c, and activation of both intrinsic and extrinsic caspase pathways. This agent also inhibited proliferation and activated apoptosis in patient-derived MM cells and neoplastic cells from patients with other hematologic malignancies. Importantly, carfilzomib showed increased efficacy compared with bortezomib and was active against bortezomib-resistant MM cell lines and samples from patients with clinical bortezomib resistance. Carfilzomib also overcame resistance to other conventional agents and acted synergistically with dexamethasone to enhance cell death. Taken together, these data provide a rationale for the clinical evaluation of carfilzomib in MM.

Description: Introduction: The ubiquitin-proteasome pathway has been validated as a therapeutic target with the approval of the small molecule proteasome inhibitor, bortezomib (VELCADE®), in multiple myeloma and non-Hodgkin lymphoma. However, the overall response rate of patients with multiple myeloma in phase III clinical trials was 43%, underscoring the need for a next generation of inhibitors with the potential for greater efficacy. Methods: PR-171 is a novel, tetrapeptide epoxomicin-related inhibitor that binds the proteasome irreversibly, and our objectives were to evaluate its activity and mechanism of action in pre-clinical models of multiple myeloma. Results: PR-171 potently bound and inhibited the chymotrypsin-like subunit of the proteasome in vitro, in cellulo, and in vivo at low concentrations. At higher concentrations, however, unlike bortezomib, which targeted the chymotrypsin-like and peptidyl-glutamyl peptide hydrolyzing activities in vivo, PR-171 also displayed significant inhibition of the trypsin-like and the peptidyl-glutamyl peptide hydrolyzing activities. PR-171-induced proteasome inhibition was associated with accumulation of polyubiquitinated substrates and pro-apoptotic Bax. Brief pulse PR-171 exposure, which simulates the in vivo pharmacokinetics of bortezomib, led to PR-171-mediated inhibition of cellular proliferation linked to induction of caspase-3-dependent apoptosis through both intrinsic (caspase-9) and extrinsic (caspase-8-dependent) pathways. Pretreatment with caspase-3, -8, and -9 inhibitors rescued the anti-proliferative effect of PR-171. Furthermore, pulse PR-171 treatment activated c-Jun-N-terminal kinase, a key-signaling molecule in proteasome inhibitor-induced apoptosis, and cleavage of poly-ADP-ribose polymerase, while abrogation of c-Jun-N-terminal kinase signaling with a dominant-negative c-Jun inhibited PR-171-induced effects. PR-171 displayed enhanced anti-proliferative activity compared to bortezomib in multiple myeloma cell lines and freshly isolated patient-derived CD138+ plasma cells, associated with enhanced phosphorylation of c-Jun-N-terminal kinase and capase-3, -8, and -9 activation. Lastly, PR-171 was a potent inhibitor of proliferation in a multiple myeloma cell line model resistant to bortezomib and in isolates from two patients, one with primary and the other with acquired bortezomib-resistance. Conclusions: These data indicate that PR-171 has enhanced activity against preclinical models of multiple myeloma, perhaps owing to its irreversible binding and subunit specificity, and provide a rationale for its translation into the clinic.

Description: Abstract 3985 Introduction: Although the combination of PLD and B improved time to progression (TTP) compared with B alone in patients (pts) with R/R MM, the overall response rate (ORR) of the regimen was only 44%, while TTP was 9.3 months. As such, strategies that build upon the efficacy of this regimen are needed. Vorinostat (V) is a histone deacetylase inhibitor that has demonstrated additive to synergistic activity with proteasome inhibitors and anthracyclines in preclinical models of MM. We therefore conducted a phase I study evaluating the safety and preliminary efficacy of V when combined with the PLD/B backbone. Patients and Methods: Pts were treated with standard doses of B and PLD (B 1.3mg/m2 on D1, 4, 8, 11, and PLD 30mg/m2 on D4) and escalating doses of V from either D4-11 or D1-14 of a 3-week cycle. Dose escalation followed a standard 3 + 3 design. Eligibility criteria included a diagnosis of relapsed or relapsed/refractory MM, absolute neutrophil count ≥1.0×109/L, platelets ≥100×109/L, creatinine clearance ≥30mL/min, and adequate hepatic and cardiac function. The primary objective was to determine the dose limiting toxicities (DLTs) and maximum tolerated dose (MTD) of the regimen and the secondary objective was to assess preliminary efficacy. Results: 32 pts were enrolled at the following dose levels: The median age was 61 (39–75) and median β2-microglobulin 3.57 mcg/mL (1.26–10.6). 69% of the pts were male, 31% female. The median time from diagnosis was 46 months (13–155) and median number of prior lines of therapy 2 (1–9). 78% of pts had received prior B, 56% PLD or doxorubicin, 91% thalidomide and/or lenalidomide, and 66% autologous and/or allogeneic stem cell transplantation. 44% (11 of 25 pts) had disease refractory to prior B-based therapy. The median number of complete cycles administered was 6 (0–15). No patients on dose level 1 or 2 suffered DLTs. One of 6 pts on dose level 3 had a DLT consisting of grade 4 systolic dysfunction in the setting of atrial flutter, which subsequently resolved. Two of 6 pts at dose level 4 experienced grade 4 thrombocytopenia in cycle 1, establishing dose level 3 as the MTD. Nine pts experienced serious adverse events at least possibly attributable to protocol therapy, including the 1 case of systolic dysfunction/atrial flutter noted above, 2 cases of nausea and vomiting with dehydration, and diarrhea, diastolic dysfunction, upper respiratory infection, syncopal episode and hypertension in 1 pt each. Grade 3 neutropenia was seen in 34% of pts (3% grade 4). 4 pts had grade 3 infections (1 attributed to protocol therapy), but no grade 4 infections were seen regardless of attribution. Grade 3 and 4 thrombocytopenia was documented in 16% and 34%, but no serious hemorrhagic events were seen. Non-hematologic toxicity at least possibly attributable to therapy included fatigue in 63% (16% grade 3, 0% grade 4). GI toxicity was common with anorexia, constipation, diarrhea, nausea and vomiting occurring in 47% (3% grade 3), 50%, 81% (16% grade 3, 3% grade 4), 78% (9% grade 3) and 50% (9% grade 3) of pts, respectively. Peripheral neuropathy at least possibly attributable to therapy was seen in 38% of pts (6% grade 3), while hand-foot syndrome was seen in 25% (9% grade 3). There were no deaths on study. Among 31 evaluable pts, the ORR using International Uniform criteria was 65% (95% confidence interval (CI): 45–81%), and the ≥very good partial remission (VGPR) rate was 29% (95% CI: 14–48%). The ORR + minimal response (MR) rate was 74% (95% CI: 55–88%). Of 14 pts with B-sensitive disease, there was 1 MR, 5 PRs and 5 VGPRs. Two PRs, 2 VGPRs and 1 complete remission (CR) were documented in 6 pts with B-naïve disease. Notably, there were 2 MRs, 4 PRs and 1 CR out of the 11 pts with B-refractory disease. Conclusions: The MTD of vorinostat when added to the PLD/bortezomib backbone is 400 mg administered daily on days 4–11. The ORR is highly promising, with responses seen in pts with bortezomib-naïve, -sensitive and -refractory disease. Although serious toxicities were infrequent, constitutional and GI side effects were highly prevalent. All together, our data support further development of this combination in pts with MM, with special attention to developing strategies and guidelines to better ameliorate toxicity. Disclosures: Voorhees: Merck: Research Funding; Celgene: Research Funding; Centocor Ortho Biotech: Research Funding; MedImmune: Consultancy; Pfizer: Research Funding. Off Label Use: Vorinostat for the treatment of relapsed and relapsed and refractory multiple myeloma. Gasparetto:Millennium Pharmaceuticals: Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees. Richards:Merck: Consultancy. Garcia:Millennium Pharmaceuticals: Speakers Bureau; Sigma-Tau: Speakers Bureau. MacLean:Novartis: Speakers Bureau. Orlowski:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Honoraria; Johnson and Johnson: Research Funding.