Resistant Multiple Myeloma Research Articles

Abstract 4424: Molecular diagnostics of drug resistant multiple myeloma cases using targeted next generation sequencing

Abstract In multiple myeloma (MM), there are two distinct genetic subtypes based on copy number alterations and translocations. About half of all MM cases are a hyperdiploid type, which is characterized by multiple trisomies of chromosomes 3, 5, 7, 9 11, 15, 19 and 21 and a lower prevalence of primary translocations involving the immunoglobulin heavy chain (IgH) locus at 14q32. The remaining cases are the non-hyperdiploid type, and chromosomes 8, 13, 14, and 16 are frequently lost. Non-hyperdiploid myeloma is strongly association with translocations of IgH alleles with various partner chromosomes, such as t(11;14)(q13;q32), t(4;14)(p16.3; q32), t(14;16)(q32;q23), t(6;14)(p21;q32) or t(14;20) (q32;q11). The copy number alterations in other chromosome regions such as 1q, 6q, 8p, and 16q occur in both subtypes. Overall, non-hyperdiploid MM is associated with worse survival than hyperdiploid MM. In this study, we sequenced all exons of 409 cancer-related genes in matched tumor and normal DNA from 5 non-hyperdiploid MM patients using a next-generation semiconductor sequencing protocol. DNA was extracted from magnetic bead-enriched bone marrow CD138 positive tumor cells from the patients and CD138 negative cells were used as matched normal cells. We detected both point mutations and copy number variations (CNVs). One individual with refractory MM (IgG λ type, ISS stage II) displayed 8 non-synonymous somatic mutations in addition to numbers of CNVs including CCND1 and RB1. We observed 2 point mutations and 3 CNVs, affecting NF-kB pathway genes: IKBKB, CYLD, IKBKE, CD79B and SYK. Although the basis of NF-kB pathway activation is only partially understood, our findings greatly expand the mechanisms by which NF-kB may be activated in this patient. We also detected several gene alterations, which may be associated with poor prognosis and poor response to chemotherapy in patients with refractory MM. Although its value should be further confirmed in larger samples, the targeted next generation sequencing is a valuable tool for high-throughput genetic testing in clinical research. Citation Format: Hiroshi Ikeda, Yasushi Sasaki, Tetsuyuki Igarashi, Yuka Aoki, Toshiaki Hayashi, Tadao Ishida, Takashi Tokino, Yasuhisa Sinomura. Molecular diagnostics of drug resistant multiple myeloma cases using targeted next generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4424. doi:10.1158/1538-7445.AM2015-4424

An Overview of Bortezomib-Induced Neurotoxicity.

The boronic acid dipeptide bortezomib, able to induce tumor cell death by degradation of key proteins, is the first proteasome inhibitor drug to enter clinical practice. It is employed as first-line treatment in relapsed or resistant multiple myeloma (MM) patients. However, bortezomib often induces a dose-limiting toxicity in the form of painful sensory neuropathy, which can mainly be reduced by subcutaneous administration or dose modification. In this review we focus on the current understanding of the pathophysiological mechanisms of bortezomib-induced neuropathy to allow further studies in animal models and humans, including analysis of clinical and pharmacogenetic aspects, to optimize the treatment regimens.

Small molecule inhibitor screen identifies synergistic activity of the bromodomain inhibitor CPI203 and bortezomib in drug resistant myeloma.

Despite significant therapeutic progress in multiple myeloma, drug resistance is uniformly inevitable and new treatments are needed. Our aim was to identify novel, efficacious small-molecule combinations for use in drug resistant multiple myeloma. A panel of 116 small molecule inhibitors was used to screen resistant myeloma cell lines for potential therapeutic targets. Agents found to have enhanced activity in the bortezomib or melphalan resistant myeloma cell lines were investigated further in combination. Synergistic combinations of interest were evaluated in primary patient cells. The overall single-agent drug sensitivity profiles were dramatically different between melphalan and bortezomib resistant cells, however, the bromodomain inhibitor, CPI203, was observed to have enhanced activity in both the bortezomib and melphalan resistant lines compared to their wild-type counterparts. The combination of bortezomib and CPI203 was found to be synergistic in both the bortezomib and melphalan resistant cell lines as well as in a primary multiple myeloma sample from a patient refractory to recent proteasome inhibitor treatment. The CPI203-bortezomib combination led to enhanced apoptosis and anti-proliferative effects. Finally, in contrast to prior reports of synergy between bortezomib and other epigenetic modifying agents, which implicated MYC downregulation or NOXA induction, our analyses suggest that CPI203-bortezomib synergy is independent of these events. Our preclinical data supports a role for the clinical investigation of the bromodomain inhibitor CPI203 combined with bortezomib or alkylating agents in resistant multiple myeloma.

Dose-adjusted Lenalidomide Combined with Low-dose Dexamethasone Rescues Older Patients with Bortezomib-resistant Multiple Myeloma.

The aim of this study was to assess the safety and efficacy of lenalidomide (Len), with the dose adjusted according to the renal function, plus low-dose dexamethasone (Dex) in older patients with bortezomib (Bor)-resistant multiple myeloma (MM). The study included 68 consecutive patients 70 years of age or older diagnosed with MM at our institute and ineligible for high-dose melphalan therapy plus autologous stem cell transplantation. Fifteen older patients with relapsed or refractory MM (RRMM) previously treated with Bor-containing regimens were treated with the combination of Len plus low-dose Dex. The median treatment duration was 12 months (range, 9 to 43 months), with all patients responding to Len plus low-dose Dex. All patients showed significant renal dysfunction between the beginning and end of treatment; however, the renal function improved in all cases. Treatment with dose-adjusted Len combined with low-dose Dex is an effective and safe therapy for older RRMM patients exhibiting renal impairment after receiving Bor-based therapies.

Myxoma virus attenuates expression of activating transcription factor 4 (ATF4) which has implications for the treatment of proteasome inhibitor-resistant multiple myeloma.

The recent development of chemotherapeutic proteasome inhibitors, such as bortezomib, has improved the outcomes of patients suffering from the plasma cell malignancy multiple myeloma. Unfortunately, many patients treated with these drugs still suffer relapsing disease due to treatment-induced upregulation of the antiapoptotic protein Mcl1. We have recently demonstrated that an oncolytic poxvirus, known as myxoma, can rapidly eliminate primary myeloma cells by inducing cellular apoptosis. The efficacy of myxoma treatment on proteasome inhibitor–relapsed or –refractory myeloma, however, remains unknown. We now demonstrate that myxoma-based elimination of myeloma is not affected by cellular resistance to proteasome inhibitors. Additionally, myxoma virus infection specifically prevents expression of Mcl1 following induction of the unfolded protein response, by blocking translation of the unfolded protein response activating transcription factor (ATF)4. These results suggest that myxoma-based oncolytic therapy represents an attractive option for myeloma patients whose disease is refractory to chemotherapeutic proteasome inhibitors due to upregulation of Mcl1.

PYK2 Inhibitors Sensitize Hypoxia-Induced Drug Resistant Multiple Myeloma Cell

Introduction: Multiple myeloma (MM) is a plasma cell malignancy, characterized by plasma cell accumulation in the bone marrow (BM) and hyperproduction of immunoglobulin IgG. Despite the implementation of novel therapies, more than 70% of MM patients relapse due to drug resistance and micro-residual disease attributed to cancer stem cells. The hypoxic nature of the BM plays a critical role in MM cells acquiring a stem cell-like phenotype, and together with cellular and acellular components of the BM microenvironment contributes to drug resistance leading to minimal-residual disease. In this study, we tested inhibition of proline-rich tyrosine kinase 2 (PYK2), using VS-4718 and VS-6063, on reversing the hypoxia-inducible stem cell-like phenotype of MM cells and sensitizing them to therapy both in vitro and in vivo.Methods: VS-4718 and VS-6063 FAK/PYK2 inhibitors were obtained from Verastem, Inc. MM cell lines (MM1s, H929 and RPMI8226) were treated with bortezomib or carfilzomib under normoxic (21% O2) or hypoxic (1% O2) conditions, in the presence or absence of the FAK/PYK2 inhibitors VS-4718 or VS-6063. MM cells were analyzed for cell proliferation by MTT assay; apoptosis was analyzed by Annexin V/PI staining and analyzed by flow cytometry; and cell signaling associated with cell proliferation and apoptosis was analyzed by western blotting.Furthermore, we have tested the effect of the PYK2 inhibitors on sensitization of bortezomib-resistant MM cells in two models in vivo. In the first model, MM1s-Luc-GFP cells were injected intravenously into SCID mice and tumors were allowed to grow for four weeks; animals were then treated with a low-dose of bortezomib (0.5mg/kg twice a week) for two weeks to induce resistance to bortezomib. The mice were divided into 6 groups treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) VS-6063 (50mg/kg) alone (4) bortezomib alone, (5) combination of VS-4718 and bortezomib, and (6) combination of VS-6063 and bortezomib, where VS-4718 and VS-6063 were administered by oral gavage twice a day every day. Mice survival was then followed for four more weeks.In the second model, H929 cells were injected subcutaneously into SCID mice. When tumors reached a mean of ~128 mm3 mice were randomized into five groups and treated with (1) vehicle, (2) VS-4718 (50mg/kg) alone, (3) bortezomib (1mg/kg) alone, (4) combination of VS-4718 and bortezomib (concurrently), and combination of VS-4718 and bortezomib (sequentially to simulate minimum residual disease). VS-4718 was administered by oral gavage twice a day and tumor growth was measured twice weekly using calipers.Results: We found that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells in both hypoxia and normoxia as single agents. Moreover, we found that hypoxia induced drug resistance to bortezomib and carfilzomib in MM cells. The combination of the two PYK2 inhibitors with either of the proteasome inhibitors reversed the hypoxia-induced resistance in MM cells and induced a synergistic effect with proteasome inhibitors on inhibition of cell proliferation, apoptosis, and cell signaling related to proliferation and apoptosis. In vivo study revealed that mice that developed MM disease with resistance to bortezomib were resensitized by treatment with the PYK2 inhibitors and showed significantly extended survival compared to mice treated with bortezomib alone. Furthermore, in mice treated with bortezomib only, tumors progressively re-grew after bortezomib treatment ended. While, in the bortezomib and VS-4718 combination treated mice and mice treated with VS-4718 in minimal residual setting (MRD) tumor grow-back (relapse) was prevented after cessation of bortezomib treatment.Conclusions: We report that FAK/PYK2 inhibitors, VS-4718 and VS-6063, decreased proliferation and increased apoptosis of MM cells as single agents. Hypoxia induced resistance to proteasome inhibitors and the PYK2 inhibitors resensitized MM cell to therapy in vitro and in vivo. Moreover, FAK/PYK2 inhibitor VS-4718 was able to prevent relapse in in vivo MM model simulating minimal residual disease. These data provide a basis for future clinical trials to sensitize relapsed/refractory MM patients to therapy by FAK/PYK2 inhibitors and their use to reduce relapse post front line treatment in an MRD setting. DisclosuresTrombino:Verastem: Employment. Padval:Verastem: Employment. Azab:Verastem: Research Funding.

PJ34, a poly(ADP-ribose) polymerase (PARP) inhibitor, reverses melphalan-resistance and inhibits repair of DNA double-strand breaks by targeting the FA/BRCA pathway in multidrug resistant multiple myeloma cell line RPMI8226/R.

There is still no ideal treatment for multidrug resistant multiple myeloma, looking for drugs which can reverse chemotherapy resistance and enhance curative effects of chemotherapy drugs becomes a problem that needs to be solved urgently. Poly(ADP-ribose) polymerase inhibitors appear to be an important tool for medical therapy of several malignancies. In the present study, we investigated the potential of the PARP-1 inhibitor PJ34, invitro, to further enhance the efficacy of the traditional chemotherapy drug melphalan in the multidrug-resistant multiple myeloma cell line RPMI8226/R. The effects of different concentrations of PJ34 and melphalan on cell proliferation were determined by the CCK-8 assay. The expressions of FA/BRCA pathway-related factors were detected by western blotting and RT-PCR. The percentage of cell apoptosis was measured with flow cytometry. DNA double-strand break (DSB) repair was quantified by γH2AX immunofluorescence. In addition, DNA damage repair at the level of the individual cell was determined by comet assay. Co-administration of PJ34 and melphalan had synergistic inhibitory effects on the proliferation of RPMI8226/R cells, suggesting more powerful antitumor activities. The apoptosis percentage also was increased more obviously by the treatment of melphalan plus PJ34. The activation of FA/BRCA pathway was inhibited by downregulation of related factors including FANCD2, BRCA2 and Rad51. PJ34 significantly increased the ratio of γH2AX-positive cells and the number of foci/cells. The comet tail rate of cells, tail length, tail moment and Olive tail moment all increased after PJ34 treatment in RPMI8226/R cells. These results indicate that PJ34 combined treatment with melphalan produces synergistic effects and reverses multidrug resistance of RPMI8226/R cells effectively. PJ34 cannot induce DNA damage directly, but it may increase the DNA damage induced by melphalan through inhibiting DNA damage repair. The suppression of FA/BRCA pathway may be the mechanism. Therefore, we suggest that PARP inhibitors may deserve future investigations as tools for medical treatment of multidrug resistant multiple myeloma.

Novel Targeting of Phospho-cMET Overcomes Drug Resistance and Induces Antitumor Activity in Multiple Myeloma

The aim of the study was to verify the hypothesis that the cMet oncogene is implicated in chemio- and novel drug resistance in multiple myeloma. We have evaluated the expression levels of cMET/phospho-cMET (p-cMET) and the activity of the novel selective p-cMET inhibitor (SU11274) in multiple myeloma cells, either sensitive (RPMI-8226 and MM.1S) or resistant (R5 and MM.1R) to anti-multiple myeloma drugs, in primary plasma cells and in multiple myeloma xenograft models. We found that resistant R5 and MM.1R cells presented with higher cMET phosphorylation, thus leading to constitutive activation of cMET-dependent signaling pathways. R5 cells exhibited a higher susceptibility to the SU11274 inhibitory effects on viability, proliferation, chemotaxis, adhesion, and to its apoptogenic effects. SU11274 was able to revert drug resistance in R5 cells. R5 but not RPMI-8226 cells displayed cMET-dependent activation of mitogen-activated protein kinase pathway. The cMET and p-cMET expression was higher on plasma cells from patients with multiple myeloma at relapse or on drug resistance than on those from patients at diagnosis, complete/partial remission, or from patients with monoclonal gammopathy of unknown significance. Viability, chemotaxis, adhesion to fibronectin or paired bone marrow stromal cells of plasma cells from relapsed or resistant patients was markedly inhibited by SU11274. Importantly, SU11274 showed higher therapeutic activity in R5- than in RPMI-8226-induced plasmocytomas. In R5 tumors, it caused apoptosis and necrosis and reverted bortezomib resistance. Our findings suggest that the cMET pathway is constitutively activated in relapsed and resistant multiple myeloma where it may also be responsible for induction of drug resistance, thus providing the preclinical rationale for targeting cMET in patients with relapsed/refractory multiple myeloma.

Blockade of the Hedgehog Signaling Pathway by the Novel Agent NVP-LDE225 Induces Differentiation, Prevents De-Differentiation and Inhibits Proliferation of Multiple Myeloma Stem Cells in Vitro

AbstractAbstract 3950Chemotherapy-resistant cancer stem cells are a major cause of relapse. It is considered that the small population of CD38++ cells which also exhibit “side population” (SP) characteristics after Hoescht 33342 staining contain chemotherapy resistant multiple myeloma (MM) stem cells. The hedgehog (Hh) signalling pathway is a key regulator of stem cell proliferation and differentiation. A range of Hh inhibitors are currently under development as therapeutics. Our aim was to determine the effect that NVP-LDE225, a novel Hh pathway inhibitor has on MM stem cells.NVP-LDE225 was kindly supplied by Novartis. Side population (SP) cells, considered to contain the MM stem cells, were detected after Hoescht 33342 staining using a BD ARIA II flow cytometer, Proliferation was monitored with CFSE tracking and viability with propidium iodide. MM cell lines RPMI8226, KMS-11, OPM2, U266 and primary MM cells were included. Expression of Hh pathway proteins PTCH1, Smo and Gli1 was determined by flow cytometry. Bone marrow samples were obtained from patients with myeloma after informed consent.CD38++ SP cells were identified in all 4 MM cell lines tested and 90% (26/29) of MM BM samples with the percentage of SP cells ranging from 0 to 9.5% of CD38++ cells. There was no correlation between %SP and %CD38++ and no significant difference in %SP during therapy. PTCH1 expression was higher on SP cells of KMS-11, OPM2 and U266 than non-SP cells (80.5% vs 51.7%) suggesting that the canonical Hh pathway was upregulated in CD38++ SP cells. PTCH1 expression varied from 0.1 – 87% on patient’s CD38++ cells. In vitro dose response curves demonstrated increased killing of plasma cell lines above 1μM NVP-LDE225. The IC50 was 4.7uM for KMS-11 SP cells and 3.4uM for KMS-11 non-SP cells. During culture of flow-sorted CD38++ SP+ and non-SP cells (RPMI 8226 and KMS11), fluctuations in %SP suggest that SP cells differentiated to non-SP cells but also that the SP phenotype is unstable. This is supportive of the concept that the stem cell compartment is in a state of flux. The presence of NVP-LED225 caused a 90% inhibition of non-SP returning to SP and induced an additional 79% differentiation of SP to non-SP KMS11 cells on day 2. Proliferation studies using CFSE tracking of sorted SP cells demonstrated a 34% reduction in proliferation after exposure to NVP-LDE225. Studies with 9 primary human bone marrow samples from patients with MM confirmed the data from cell lines demonstrating that differentiation was significantly induced (t=2.78; p<0.0275) and de-differentiation was blocked.The studies confirm there is flux, to and from the SP and presumably MM stem cell compartment, that the canonical Hh pathway is upregulated in MM SP cells, and that the Hh pathway inhibitor NVP-LDE225 can induce SP differentiation, block de-differentiation, inhibit SP proliferation and at high concentrations is cytotoxic to all cells including MM stem cells. NVP-LDE25 could synergise with other maintenance therapy to reduce drug resistance by inducing the differentiation of chemo-resistant MM stem cells. Disclosures:No relevant conflicts of interest to declare.

Thalidomide: Chemistry, Therapeutic Potential and Oxidative Stress Induced Teratogenicity

Thalidomide and its one analogue, lenalidomide (CC5103 or revlimid) are recently approved for the treatment of multiple myeloma. Multiple myeloma is characterized by an overproduction of malignant plasma cells in the bone marrow. The journey of thalidomide was started in 1956 when it was marketed as a non-barbiturate sedative agent. It was considered as a "wonder drug" that provided safe and sound sleep and hence, used to cure morning sickness in pregnant women. Later, in 1961, it was withdrawn from the world market due to its serious side effects, i.e., teratogenic activity. However, the recent decade has witnessed a true renaissance in interest in its broad biological activity. In particular, thalidomide was reevaluated and attracted significant attention due to its selective inhibitory activity of tumor necrosis factor-α (TNF-α), which is a clinically important activity against serious diseases such as rheumatoid arthritis, Crohn's disease, leprosy, AIDS, and various cancers. The comeback of thalidomide to the legitimate status of a marketed drug came in 1998 when it received FDA approval for the treatment of erythema nodosum leprosum (ENL). Recently, the drug has got FDA approval for the treatment of multiple myeloma. In the last few years, number of thalidomide analogues have been synthesized and are in clinical development as a class of immunomodulatory drugs. Among these, lenalidomide is more potent than thalidomide, and is also non-neurotoxic. It was shown in vitro studies to induce apoptosis or arrest growth even in resistant multiple myeloma cell lines, decrease binding of the cells to bone marrow stromal cells, and stimulate host natural killer cell immunity. It also inhibits tumour growth and decreases angiogenesis. Earlier reviews have described the pharmacological aspects of thalidomide and a review has focused only on synthetic aspect of thalidomide. However, review focusing on chemistry and metabolism and mechanism of biological activity is still lacking. In this review, we will concisely describe the therapeutic aspects, metabolism and synthesis of thalidomide.

Bortezomib salvage followed by a Phase I/II study of bortezomib plus high‐dose melphalan and tandem autologous transplantation for patients with primary resistant myeloma

We conducted a Phase 1/2 study of bortezomib administered in combination with high-dose melphalan followed by tandem autologous transplants in patients with primary resistant multiple myeloma. Thirty patients received two cycles of salvage bortezomib followed by stem cell mobilization with granulocyte colony-stimulating factor and harvest. Melphalan 100 mg/m(2) per day on two consecutive days was administered, immediately followed by one dose of bortezomib (dose escalation) and stem cell infusion. The median beta 2-microglobulin was 4·35 mg/l (range: 1·8-11·4); albumin was 37 g/l (range: 3·1-4·9); high-risk karyotypes were noted in 45% of patients. The maximum planned dose of bortezomib at 1·3 mg/m(2) was well tolerated and a formal maximum tolerated dose was not determined. The peak of best overall response (≥partial response) and complete response rates after tandem transplants were 84% and 36%, respectively. With a median follow-up of 48 months, the median progression-free survival was 15 [95% confidence interval (CI): 11-21] months and the median overall survival was 35 (95% CI: 22-43) months. Correlative studies demonstrated decreased expression of BRCA2 (P = 0·0072) and FANCF (P = 0·0458) mRNA following bortezomib treatment. Bortezomib combined with high-dose melphalan is a well-tolerated conditioning with some activity in patients with resistant myeloma.

Efficacy and Safety of Lenalidomide in the Treatment of Multiple Myeloma (MM) Patients: a Preliminary Report of Polish Myeloma Group

Abstract 5125The aim of the multi-centre observational study was to evaluate the efficacy and safety of lenalidomide therapy in patients with resistant or relapsed multiple myeloma as well in maintenance treatment.The study involved 84 patients, 47 women and 37 men, aged 38–84, mean age 57.8 years; in Stage I, II and III according to Durie - Salmon staging system there were 6, 28 and 50 patients respectively; 12 patients developed renal failure. Fourty-eight patients had IgG myeloma, 26 – IgA, 6 patients were diagnosed with light chain disease, 2 – with non-secretory myeloma and 56 with kappa light chain disease. Prior to lenalidomide therapy the patients had received 1–8 treatment lines (mean:3). In 19 patients (22.6%), who were in disease remission (including CR 2, VGPR 6 and PR 11) lenalidomide was instituted due to polyneuropathy (Cohort 1, C1), among the remaining patients, on the onset of lenalidomide therapy 27 patients (32.1%, Cohort 2, C2) were diagnosed with resistant multiple myeloma and 38 patients (45.3%, Cohort 3, C3) had relapse of the disease. Lenalidomide was administered at the dose of 25mg p.o, on days 1–21 (15 pts in C1 and 54 in C2 and C3), at the dose of 15mg, 10mg or 5 mg for 21 days (1 and 2 patients in C2 and 1, 2 and 1 patient in C3 respectively) and dexamethasone at the dose of 20 mg on days 1–4, 8–11 and 17–20. Four patients with C1 and 2 with C2 received only lenalidomide at the dose of 25 mg for 21 days; individual patients from group C2 were administered lenalidomide with bortezomib or bendamustin and dexamethasone. The cycles were repeated every 28 days. All the patients were administered aspirin 75mg in the prophylaxis of deep vein thrombosis.Therapeutic response was evaluated on the basis of modified criteria of the European Group for Blood and Marrow Transplantation (EPMT) in patients who had completed at least 3 therapeutic cycles.In patients who were administered lenalidomide as a maintenance therapy, there were no cases of disease progression, and the percentage of CR and VGPR increased (from 42% to 68.4%), in the group of resistant patients the percentage of therapeutic response was 44.4% and in the group of relapsed myeloma it was 44.7%. The analysis of response rates in patients with maintenance therapy, resistant and relapsed MM is shown in Table 1.Table 1Response rate analyzed by disease status.C1 n=19 maintenance therapyC2, n=27 resistantC3, n=38 relapsedCR3 (15.7%)1 (2.6%)VGPR10 (52.6%)2 (7.4%)3 (7.8%)PR6 (31.5%)10 (37.0%)13 (34,2%)SD12 (44.4%)13 (34.2%)PD3 (11.1%)8 (20.9%)During the observation period there were three deaths: 2 patients died due to disease progression, 1 patient due to pulmonary thrombosis. Other undesired effects included neutropenia and thrombocytopenia (II and III grade), and infections. Summary:Lenalidomide with dexamethasone is an effective and well tolerated both in maintenance therapy as well as in resistant and relapsed patients. Disclosures:No relevant conflicts of interest to declare.

Targeting the Over-Expressed Transcription Factor Yin-Yang 1 (YY1) Sensitizes Resistant Multiple Myeloma (MM) Cell Lines to Apoptosis by Bortezomib or Melphalan,

Abstract 3991The transcription factor Yin-Yang 1 (YY1) is a multi-functional DNA-binding protein which can activate, repress, or initiate transcription depending on the context in which it binds. We have previously reported that YY1 is a repressor for the death receptors Fas and DR5 as well as it regulates both chemo- and immuno- resistance in many tumors such as prostate carcinoma and lymphoma. YY1 has also been reported to be a prognostic factor for some cancers. Due to the inherent resistance of multiple myeloma (MM) cell lines to various cytotoxic drugs, we examined the expression levels of YY1 in both MM cell lines and bone marrow (BM)-derived MM tissues. Analysis for YY1 was performed by immunohistochemistry (IHC) and western blot. We found that, compared to normal peripheral blood mononuclear cells and normal bone marrow, the expression of YY1 was significantly upregulated in both the cell lines and patient-derived MM tissues. The expression of YY1 was distributed in both the cytoplasm and the nucleus. Further, the intensity and frequency of cells expressing YY1 in the nuclei of MM tissues were significantly higher among patients with progressive disease as compared to patients with stable disease. In order to ascertain the role of YY1 in the maintenance of drug resistance in MM, we hypothesized that, due to the overexpression of YY1 in MM, inhibition of YY1 expression and activity should lower the threshold of resistance and render the MM cells more sensitive to drug-induced apoptosis. This hypothesis was tested using the MM U266 cell line and the drugs bortezomib and melphalan as models. The inhibition of YY1 was done by knocking down YY1 following transfection with YY1 siRNA. Analysis of the transfected cells with YY1 siRNA, but not with control siRNA, inhibited YY1 expression as analyzed by IHC and western blot. The U266 cells were first transfected with YY1 siRNA or control siRNA for 48 hours and then treated with various concentrations of bortezomib (2.5 and 5.0 nM) or melphalan (10 and 20 μM) that were not toxic to the cells for 48 hours. The cells were analyzed for apoptosis by activation of the effector caspase 3. The findings demonstrate that YY1/siRNA-treated cells were sensitized to apoptosis by bortezomib or melphalan. Analysis of the underlying mechanism by which the inhibition of YY1 sensitizes the MM cells via regulation of the apoptotic pathways will be presented. We are also currently testing specific chemical inhibitors for YY1 that are being tested both in vitro and pre-clinically in mice.The present findings demonstrate that YY1 is a potential target for therapeutic intervention and the combination of YY1 inhibitors with non-toxic doses of bortezomib, melphalan or other cytotoxic drugs may be beneficial for the treatment of MM patients who are no longer responding to current treatments. Further, we propose that, based on our findings that the level and nuclear localization of YY1 dictates disease progression in a subset of patients, inhibition of YY1 with treatment may reduce the frequency of patients with progressive disease. *Contributed equally Disclosures:No relevant conflicts of interest to declare.

Sensitizing human multiple myeloma cells to the proteasome inhibitor bortezomib by novel curcumin analogs

The proteasome plays a vital role in the degradation of proteins involved in several pathways including the cell cycle, cellular proliferation and apoptosis and is a validated target in cancer treatment. Bortezomib (Velcade®, PS-341) is the first US FDA approved proteasome inhibitor anticancer drug used in the treatment of refractory multiple myeloma. In spite of its improved efficacy compared to alternative therapies, about 60% of patients do not respond to bortezomib due to the emergence of resistance. We hypothesized that novel small molecules could enhance the proteasome-inhibitory and anticancer activities of bortezomib in resistant multiple myeloma cells in vitro and in vivo. The dietary polyphenol curcumin has been shown to exert anti-cancer activity in several cancer cell lines, but the effects of curcumin in solid tumors have been modest primarily due to poor water solubility and poor bioavailability in tissues remote from the gastrointestinal tract. Here we show that the water-soluble analog of curcumin #12, but not curcumin, in combination with bortezomib could enhance the proteasome-inhibitory effect in multiple myeloma cells. Furthermore, the sensitivity of the myeloma cells to cytotoxic killing in the presence of otherwise sublethal concentrations of bortezomib was enhanced by incubation with the curcumin analog #12. These findings justify further investigation into those combinations that may yield potential therapeutic benefit.

Apoptin Induced Tumour Specific Killing Via Protein Kinase C Isoforms: A Potential Therapeutic Strategy In Plasma Cell Dyscrasias

AbstractAbstract 5039There is mounting evidence that malignant cells have an intrinsic ability to prevent apoptosis. In the present study we provide evidence that the ectopic expression of Apoptin can restore the failing apoptosis program in myeloma cells via protein kinase C b (PKCb) and overcome intrinsic or acquired resistance to cell death. Apoptin (VP3), a chicken anemia virus (CAV)-derived protein has been shown to possess tumor specific cytotoxicity; its expression induces apoptosis in human tumor and transformed cells but there is little or no cytotoxic effect in normal human cells or cell lines derived from different tissues including peripheral blood mononuclear cells, fibroblast and epithelial cells. Several studies have shown that the tumor specific killing of Apoptin correlates with its phosphorylation and its subcellular localization. In cancer cells, Apoptin is localized in the nucleus and is phosphorylated on threonine108 by an as yet unknown kinase, whereas in normal cells Apoptin is detected in the cytoplasm and is essentially unphosphorylated. We developed a lentiviral vector encoding a GFP-Apoptin fusion gene (LV-GFP-AP), which delivers the Apoptin gene efficiently to haematopoietic cells. Apoptin significantly and selectively killed a number of leukemia cell lines including K562, HL60, U937, KG1 and NB4. In particular, the dexamethasone resistant multiple myeloma cell line MM1.R and the dexamethasone sensitive cell line MM1.S were efficiently killed by Apoptin. In contrast normal CD34+ cells were not killed and maintained their differentiation potential in multilineage colony formation assays.In addition, we showed that the dexamethasone resistant MM1.R cells were considerably more susceptible to Apoptin induced cell death than the parental matched MM1.S cells. This correlated with increased phosphorylation and activation of the Apoptin protein in MM1.R cells. Expression profiling of MM1.R and MM1.S cells identified a number of differentially expressed kinases. PKCb was over-expressed 9 fold in MM1.R cells and we showed, by immunoprecipitation and in vivo kinase studies, that this kinase was responsible for Apoptin phosphorylation. Analysis of the Apoptin amino acid sequence for potential phosphorylation sites indicated seven putative phosphorylation sites corresponding to the PKC kinase consensus motifs (S/TXK/R or S/TXXK/R). These sites included Thr-108, which has been previously shown to be phosphorylated in tumor cells, but not in normal cells. In vitro studies showed that recombinant Apoptin protein was phosphorylated by recombinant GST-PKCb protein at the Thr-108 site. Addition of a PKCb specific inhibitor resulted in diminished Apoptin phosphorylation whilst an unrelated inhibitor had no such effect. Furthermore, shRNA knockdown or drug mediated inhibition of PKCb in vivo significantly reduced Apoptin phosphorylation. Finally, we found that Apoptin mediated cell death proceeded via the up-regulation of PKCb, activation of caspase-9/3, cleavage of the PKCd catalytic domain and down-regulation of MERTK and AKT protein kinases. Collectively these results demonstrate a novel pathway for Apoptin activation involving PKCb and PKCd.Our results show that Apoptin is able to effectively eliminate multiple myeloma cells which have become resistant to dexamethasone. In addition, this study has led to the identification of tumor specific cellular targets such as PKCb, whose modulation by shRNAs and small molecule drugs can induce strong anti-myeloma effects. Importantly, the evidence from our data suggests that protein kinase C inhibitors may have an important therapeutic role in plasma cell neoplasia. Disclosures:No relevant conflicts of interest to declare.

341 Targeting NEK2 kinase in drug resistant multiple myeloma with small molecule inhibitors

The epidermal growth factor receptor (EGFR) is a validated target in head and neck squamous cell carcinoma (HNSCC). In recurrent and/or metastatic (R/M) HNSCC, resistance to anti-EGFR therapy inevitably occurs. Downstream activation of the PI3K/Akt/mTOR pathway is an established resistance mechanism. Concurrent mTOR blockade may improve efficacy of anti-EGFR therapy.Erlotinib 150 mg daily and temsirolimus 15 mg weekly were administered to patients with platinum-refractory R/M HNSCC and ECOG performance status 0–2. The primary endpoint was progression-free survival (PFS). Correlative studies determined PIK3CA and HRAS mutation status; p16, EGFR, pS6K, pAkt and PTEN expression; and pre- and post-treatment plasma levels of 20 immunomodulatory cytokines.Twelve patients enrolled; six withdrew within 6 weeks due to toxicity or death, prompting early closure of the trial. Grade ⩾ 3 toxicities included fatigue, diarrhea, gastrostomy tube infection, peritonitis, pneumonia, dyspnea, and HN edema. Median PFS was 1.9 months. Median overall survival was 4.0 months. Six/12 tumors were p16(+), 9/11 lacked measurable PTEN expression, and 1/12 harbored a PIK3CA mutation. On exploratory analysis, high baseline plasma VEGF and interferon-gamma levels marginally associated with tumor progression.The combination of erlotinib and temsirolimus was poorly tolerated. Low prevalence of PTEN expression and 8% incidence of PIK3CA mutations indicate biological relevance of this pathway in R/M disease. Investigation of more tolerable combinations of EGFR and PI3K/Akt/mTOR pathway inhibitors in selected HNSCC patients is warranted.

A phase II trial of sorafenib in patients with relapsing and resistant multiple myeloma (MM) previously treated with bortezomib (S0434)

e19517 Background: The multikinase inhibitor sorafenib targets several serine/threonine and receptor tyrosine kinases by blocking RAF kinase, a critical component of the RAF/MEK/ERK signaling pathway regulated by the Ras oncogene, which is mutated both in primary patient samples and in human MM lines (35–50%). As the frequency of these mutations increases with advancing disease and increasing drug resistance, inhibition of the RAF/MEK/ERK signaling pathway, as well the angiogenic VEGFR-2/PDGFR beta cascade by sorafenib may be a useful new approach for the treatment of MM. Methods: SWOG evaluated the effect of sorafenib as a single agent in relapsed/refractory MM patients. In this phase II study we assessed response rate, overall (OS) and progression-free survival (PFS) as well as toxicities associated with this treatment. Twenty-three heavily pretreated MM patients were enrolled in the study. Sorafenib was started at oral dose of 400 mg daily until progression or toxicity. This dose was based on the label for metastatic renal cell carcinoma. Results: The study was closed as planned due to lack of efficacy in first 18 patients who were assessable for toxicity and response. Three patients experienced Grade 4 toxicity consisting of thrombocytopenia, anemia and renal failure. 8 cases suffered Grade 3 toxicities including thrombocytopenia, neutropenia, anemia, hand-foot syndrome, diarrhea and dyspnea. No responses were observed. 3 patients had stable disease (2.4–15.9 months) and the remainder progressed. Median PFS is one month, and OS at 12 months is 50%. Conclusions: Thus, single agent sorafenib did not show activity in this group of heavily pretreated MM patients previously exposed to bortezomib. As the frequency of RAS oncogene mutations increases resulting in resistance to traditional chemotherapeutic agents as well as possibly supporting cytokine resistance to immune modulators, sorafenib might have a supportive role in combination therapy with bortezomib, lenalidomide or everolimus in relapsed/refractory MM which is currently being evaluated in ongoing studies. No significant financial relationships to disclose.

A novel myeloma cell line identified for multidrug resistant study

Objective To find out how to overcome resistance during multiple myeloma (MM) treatment through establishing a multidrug resistant human multiple myeloma cell line and investigating its biological features.

5HPP-33, a Novel Tubulin-Polymerization Inhibitor Derived from Thalidomide, Directly Inhibits Proliferation and Induces Apoptosis of Human Multiple Myeloma Cells.

Multiple myeloma is an incurable hematological malignancy despite the use of high-dose chemotherapy with hematopoietic stem cell transplantation. In addition, repeated episode of relapse may lead to refractory or resistant multiple myeloma; therefore, novel therapeutic approaches are desired in clinical settings. Recently, thalidomide has been introduced in the treatment of myeloma, and many clinical trials have since confirmed its efficacy in patients with relapsed, refractory and newly diagnosed multiple myeloma. Multiple mechanisms have been proposed for the anti-myeloma activity of thalidomide; however, its precise mechanism of action is still unclear, because thalidomide rapidly undergoes spontaneous, nonenzymatic, hydrolytic cleavage to numerous metabolites in vivo. In addition, side effects and teratogenic potential of thalidomide have often prevented its use in the direct treatment of myeloma. To address the exact anti-myeloma effect of thalidomide and develop the new derivatives without causing teratogenic effects, we have performed structural development studies of thalidomide and obtained various analogues with specific molecular properties. Among these derivatives, we report here for the first time that 2-(2,6-Diisopropylphenyl)-5-hydroxy-1H-isoindole-1,3-dione (5HPP-33) has most potent anti-myeloma effect with tubulin-polymerization-inhibiting activity. 5HPP-33 inhibited cellular growth of myeloma cell lines (RPMI8226, IM9, U266) and freshly isolated myeloma cells from patients in dose (0–50 μM)- and time (0–24 h)-dependent manners with an IC50 between 1–10 μM. In contrast, thalidomide itself did not inhibit cellular growth of RPMI8226 cells except exposure with high concentration (100 μM). Cultivation with 10 μM 5HPP-33 induces G2/M phase cell cycle arrest, and a strong induction of apoptosis 3 h after treatment. Immunofluorescent staining of RPMI8226 cells with anti-tubulin antibody revealed that 10 μM 5HPP-33 inhibits tubulin-polymerization. Induction of apoptosis was also confirmed in terms of both morphological changes and DNA ladder formation. Treatment with 5HPP-33 induced caspase-3 activity and PARP cleavage. Tubulin polymerization assay using microtubule protein from porcine brain revealed that 5HPP-33 showed potent tubulin-polymerization-inhibiting activity with an IC50 of 8.1 μM, comparable to that of known tubulin-polymerization inhibitors, rhizoxin and colchicines. In addition, its activity was more potent than a known thalidomide metabolite, 5-hydroxythalidomide. Interestingly, the structural requirement for activity was critical, because other analogues and derivatives of 5HPP-33 showed only slight tubulin-polimerization-inhibiting activity. To evaluate the effects of 5HPP-33 in vivo, we are currently doing the experiments to clarify the potency of tumor reduction using RPMI8226-transplanted NOD/SCID mice model. In conclusion, a novel tubulin-polymerization inhibitor, 5HPP-33, directly inhibits proliferation and induces apoptosis of myeloma cells in vitro and in vivo. In addition, one possible mechanism of growth inhibition by thalidomide might be its tubulin-polymerization inhibition activity in vivo. Taken together, 5HPP-33 is one of the promising candidates for the new therapeutic agent of multiple myeloma.

Targeting Therapy for Resistant Multiple Myeloma with a Novel Inhibitor of NF-KB, NPI-1387.

Transcription factor NF-KB is linked to growth and survival of multiple myeloma (MM)cells; blockade of NF-KB activity is therefore an attractive therapeutic strategy. Here we describe NPI-1387, a potent inhibitor of NF-KB activation and its effects on MM cells, including those resistant to conventional agents dexamethasone or doxorubicin. Cell-based assays were used to screen a library of 200 semi-synthetic analogs derived from the pimarane diterpene, Acanthoic acid. Among these analogs, NPI-1387 inhibited LPS-induced TNF-A synthesis in the murine macrophage-like RAW 264.7 cells most potently. Importantly, NPI-1387 reduced TNF-A-induced NF-KB activation in a HEK293 NF-KB/luciferase reporter cell line. Therefore additional studies were initiated to define the biological activities in MM. Treatment of MM cells lines (MM.1S, MM.1R, OCI-My5, OPM1, Dox-40) with NPI-1347 for 48h induces a dose-dependent significant (P < 0.004) decrease in cell viability in all cell lines at pharmacologically achievable concentrations (IC50 range 25–40 micromolar). To determine whether NPI-1387-decreased cell viability is due to apoptosis, various MM cell lines were treated at their respective IC50 for 48h; harvested; and analyzed for apoptosis. NPI-1387 triggered significant apoptosis in these cells, as measured by a marked increase in nuclear condensation reflected by dense pattern of DAPI stain under phase contrast microscopy. In contrast, untreated control cells exhibited homogeneous and intact nuclei. Besides nuclear condensation, NPI-1387 triggered proteolytic cleavage of poly (ADP ribose) polymerase (PARP), a hallmark of apoptosis. Examination of purified patient MM cells demonstrated similar results. Notably, NPI-1387 decreases the viability of cells obtained from Bortezomib-refractory MM patient. In contrast, no significant toxicity of NPI-1387 was observed against peripheral blood mononuclear cells from normal healthy donors or CD138− MM patient cells. Moreover, NPI-1387 does not affect the viability of MM patient-derived bone marrow stromal cells (BMSCs). Genetic and biochemical evidence indicates that apoptosis proceeds by two major cell death pathways: an intrinsic pathway that involves mitochondrial membrane permeabilization and release of several apoptogenic factors, followed by caspase-9 activation; and an extrinsic apoptotic signaling pathway that occurs via caspase-8 activation. Both caspase-8 and caspase-9 activate downstream caspase-3. We therefore next examined whether NPI-1387 triggers extrinsic or intrinsic apoptotic signaling pathways. Our results show that NPI-1387 (25 micromolar) induces activation of caspase-8, and caspase-9, followed by caspase-3 cleavage. These data suggest that NPI-1387-triggered MM cell apoptosis predominantly proceeds via caspase-8/caspase-9>>>>caspase-3 signaling pathway. Together, these findings provide the rationale for clinical evaluation of NPI-1387 to induce MM cell killing, overcome drug-resistance, and improve patient outcome in MM.