ALBERT

Description: Recently, it has been demonstrated that the proapoptotic protein BIM showed a deletion polymorphism at exon 3 in eastern Asian population, and some CML patients with the BIM deletion polymorphism are resistant to imatinib treatment (Ng et al. Nature Medicine, 2012). More recently, a BIM single nucleotide polymorphism (SNP) at exon 8 (c465C〉T) has also been found in French CML patients and this SNP is associated with not only imatinib resistance but also the presence of BCR-ABL mutations (Mahon et al. ASH abstract, 2012). We aimed to investigate a possible association between such genetic variations of BIM and clinical manifestation in Japanese CML patients who experienced undetectable minimal residual disease (UMRD: so-called CMR4.5). In this study, we newly analyzed BIM SNP (c465C〉T) in 47 CML-UMRD patients with known BIM deletion polymorphism status (Katagiri et al. Br J Haematol, 2013). Twenty normal subjects were used as controls. The frequency of either BIM SNP at exon 8 or BIM deletion polymorphism did not deviate from the normal subjects in the Japanese population (P = 0.7597 and P = 0.2880, respectively). None of the subjects showed both BIM SNP at exon 8 and BIM deletion polymorphism concomitantly. We then compared the clinical features among 3 CML-UMRD groups: patients with BIM SNP, patients with BIM deletion polymorphism, and patients who showed neither BIM SNP nor BIM deletion polymorphism (no genetic variations). The frequency of CML patients who maintained 400 mg imatinib dose until stopping was significantly higher in those without genetic variations than in those with BIM SNP or BIM deletion polymorphism (P = 0.0002). Moreover, the frequency of CML patients who switched to second tyrosine kinase inhibitors (2nd TKIs) was significantly higher in those with BIM SNP or BIM deletion polymorphism than in those without such polymorphisms (P = 0.0055).Number of CML patientsMaintained IM 400 mgChange of imatinib dose2nd TKIs switchingBIM SNP (c465C〉T)11/474/115/112/11BIM deletion polymorphism6/471/63/62/6BIM SNP or deletion polymorphism17/475/178/174/17No BIM genetic variationss30/4725/305/300/30 This is apparently the first study to circumstantiate the BIM genetic variants in Japanese CML patients with UMPD. Although the number of patients is small, our results suggest that CML patients without BIM deletion polymorphism/SNP could be maintained under standard imatinib dose without switching to 2nd TKIs, and thereby, have a possibility to stop TKIs therapy. Disclosures: Ohyashiki: Norvartis KK: Research Funding, Speakers Bureau; Bristol Meyer Squibe KK: Research Funding, Speakers Bureau.

Description: Introduction: The BCR-ABL1 fusion gene is a causative oncogene in chronic myeloid leukemia (CML) and 30-50% of acute lymphoblastic leukemia cases. Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations and become resistant to ABL TKI therapy. In particular, the ABL kinase domain mutation T315I is resistant to imatinib and second-generation ABL TKIs (e.g., nilotinib, dasatinib, and bostinib). Accordingly, this mutation is often found in patients with TKI-resistant disease. A third-generation ABL TKI, ponatinib, was recently developed. Ponatinib is a potent oral tyrosine kinase inhibitor that affects both unmutated and mutated BCR-ABL; it is effective against T315I mutant cells and has been approved for TKI-resistant or intolerant CML and Ph-positive ALL patients.Recently, the vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib was found to exhibit anti-leukemic activity against T315I-mutant disease. Axitinib is an orally active and potent TKI of VEGFRs 1, 2, and 3 and inhibits BCR-ABL1, especially the T315I variant, via a distinct binding conformation. Materials and methods: In this study, we investigated whether axitinib could suppress ponatinib-resistant compound mutant cells harboring the T315I mutation and primary samples. Results: A 72 h axitinib treatment inhibited the growth of Ba/F3 T315I cells (Figure 1A). Immunoblot analysis of axitinib-treated cells revealed dose-dependent decreases in BCR-ABL, the downstream molecule CrkL, and ribosomal S6 protein phosphorylation and increases in caspase 3 and Poly (ADP-ribose) polymerase (PARP) activity. Ponatinib and axitinib also induced apoptosis, significantly increased caspase activity, and reduced Akt activity. In contrast, clinically available concentrations of axitinib did not inhibit the growth of ponatinib-resistant Ba/F3 cells. Immunoblot analysis revealed that BCR-ABL, Crk-L, and S6 kinase phosphorylation were not inhibited by axitinib or ponatinib. Similarly, no increase in caspase activity or decrease in Akt activity was observed following axitinib treatment, and neither ponatinib nor axitinib affected apoptosis in these cells. We next evaluated primary T315I-mutant and ponatinib-resistant compound mutant samples. Axitinib potently inhibited the growth of T315I mutant primary cells in a dose-dependent manner. Immunoblot analysis further revealed reduced Crk-L and S6 kinase phosphorylation after axitinib or ponatinib treatment. In contrast, the growth of ponatinib-resistant primary cells was not affected by ponatinib or axitinib. Immunoblotting revealed that neither ponatinib nor axitinib affected the phosphorylation of Crk-L and S6 kinase in ponatinib-resistant cells. Conclusion: In CML, ABL TKI resistance is frequently caused by ABL kinase domain mutations. The T315I mutation is resistant to all ABL TKIs except ponatinib. Although axitinib, which is currently being investigated for efficacy in patients with Ph-positive T315I-mutant leukemia, induced apoptosis in T315I-mutant cells, it was ineffective against cells with a compound mutation including T315I. Current evidence to direct the management of ABL TKI-resistant disease, particularly those harboring T315I and compound mutations, is limited. New molecular-targeted drugs and an understanding of ABL TKI resistance mechanisms are required to apply an appropriate therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Description: Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with BCR/ABL-positive leukemia. MK-0457 is a small molecule inhibitor of the Aurora kinase family with potent cross-reactivity towards wild type and T315I BCR/ABL and FLT-3, and the compound has shown activity in preclinical models of solid tumors and FLT-3 driven leukemia. MK-0457 is presently being evaluated in a clinical trial in chronic myelogenous leukemia (CML) patients with imatinib resistance such as T315I mutation, but the mechanism of MK-0457 is not fully evaluated. In this study, the gene expression profiles of CML cell line, K562, exposed to imatinib or MK-0457 were analyzed and compared. When their gene expression profiles were compared, 937 genes in imatinib and 895 genes in MK-0457 were increased and 625 genes were overlapped. In contrast, 597 genes in imatinib and 537 genes in MK-0457 were decreased and 396 genes were overlapped. These down regulated genes included heat shock proteins (HSPs) suggesting these results may relate to the BCR/ABL stability. Next, we examined the intracellular signaling of MK-0457 in BCR/ABL positive cells with T315I mutation by using BAF-3 p185 BCR/ABL (p185) and BAF-3 BCR/ABL T315I (T315I) cell lines. MK-0457 potently induced apoptosis of p185 cells and T315I cells in 72 hours treatment. IC50 of MK-0457 was 100 nM (p185) and 250 nM (T315I). We found that caspase 3, and poly (ADP-ribose) polymerase (PARP) were activated and BCR/ABL phosphorylation was reduced after MK-0457 treatment in a dose dependent manner. It has been reported that histone deacetylase inhibitor (e.g., vorinostat) also depleted BCR/ABL, as well as induced apoptosis and sensitized BCR/ABL-expressing leukemia cells to apoptosis induced by imatinib. These data suggest that MK-0457 may be synergistic with histone deacetylase inhibitors in light of recent publications that have shown that vorinostat induces acetylation of the heat shock protein (HSP), depletes the HPS90 client proteins, including BCR/ABL, and enhances the imatinib-induced apoptosis in BCR/ABL-expressing cells. We found that combination of MK-0457 and vorinostat synergistically increased the apoptosis of p185 cells and T315I cells in 72 hours treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We examined the intracellular signaling by using these cell lines. Phosphorylation of BCR/ABL, signal-transducing activators of transcription 5 (STAT5), one of the src kinase, Lyn, Crk-L were reduced after treatment with MK-0457 + vorinostat. We also found that cyclin D1 was decreased after MK-0457 or vorinostat and MK-0457 treatment. Histone H4 is also acetylated after vorinostat treatment, but phosphorylation of BCR/ABL was not reduced directly. We evaluated the activity of MK-0457 and vorinostat in primary Ph positive acute lymphoblastic leukemia cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 72 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the extent of apoptosis in primary T315I cells. This study provides further insight into the mechanism of action of MK-0457 in wild type and T315I mutant BCR-ABL cells and provides mechanistic rational for the synergistic induction of apoptosis observed for the combination of MK-0457 + vorinostat.

Description: Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy (e.g. imatinib, nilotinib and dasatinib) has improved the survival of Ph-positive leukemia patients. However, despite the impressive efficacy of these agents, disease relapse has been observed in clinically. Mutations in the BCR-ABL kinase domain can cause of ABL TKI resistance. In particular, one of the BCR-ABL kinase domain mutations (e.g. T315I) is associated with a high level of resistance to all available ABL TKIs. Ponatinib (formally, AP24534) is a multi-target TKI. Recently, in the PACE (Ponatinib Ph+ acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML) Evaluation) trial, ponatinib showed significant efficacy against Ph-positive leukemia in patients with multi-resistant T315I mutations. However, in some patients, especially those with Ph-positive ALL, ponatinib resistant clones were identified. Omacetaxine mepesuccinate, formally known as homoharringtonine is a natural alkaloid obtained from various Cephalotaxus species. Omacetaxine is a first-in-class cephalotaxine in clinical development as anti-leukemic therapy. Omacetaxine acts by binding to the A-site cleft of ribosomes and thereby transiently inhibits protein synthesis. Omacetaxine was approved for the treatment adult patients with chronic or the accelerated phase of CML resistant to other therapies. We investigated the efficacy of omacetaxine against ponatinib resistant Ph-positive cells. Ba/F3 ponatinib resistant cells (Ba/F3 ponatinib-R) have three BCR-ABL point mutations (Y253H, E255K and T315I: data not shown). With 72 h omacetaxine treatment, the cell growth of Ba/F3 ponatinib-R and Ph-positive ALL cell line was significantly reduced even at a low concentration and it is also effective to the other hematological malignancies such as acute myeloid leukemia. In contrast, Ba/F3 ponatinib-R was resistant to ponatinib. With 48 h treatment, omacetaxine dependent apoptosis was increased. Although anti-apoptotic proteins were not increased in this cell line compared to parental cells, as compound mutations such as E255V/T315I confer high-level resistance to ponatinib, these three point mutant was associated with vitro resistance to ponatinib. We also examined intracellular signaling. The phosphorylations of BCR-ABL and a down-stream molecule, Crk-L, were decreased. Protein expressions of BCR-ABL and Crk-L were also decreased. However, caspase-3 and cleaved Poly (ADP-ribose) polymerase (PARP) levels were significantly increased in low concentration. In a previous study, omacetaxine was shown to induce apoptosis in leukemic cells due to a selective decrease in short-lived proteins. We found that omacetaxine reduced the expression of BCR-ABL and heat shock protein 90(HSP90) which is stabilize BCR-ABL protein. We also found that omacetaxine reduced the expression of anti-apoptotic protein, Bcl-2. The protein expression of c-myc was also reduced. We next examined a ponatinib resistant primary Ph+ ALL and chronic phase CML samples. The ponatinib resistant primary cells have several BCR-ABL point mutations (e.g. Q252H, E255K/V, and T315I). We found the growth of primary cells was resistant to ponatinib but to be reduced after omacetaxine treatment and similar signaling events were occurred in OM-treated primary ALL cells. Omacetaxine is an inhibitor of protein synthesis. Because omacetaxine inhibits the BCR-ABL, Bcl-2 and HSP90 pathways in BCR-ABL positive leukemia cells through reduced the levels of these proteins, Omacetaxine has anti-tumor activity and promotes apoptosis. Our findings suggest that omacetaxine may benefit patients with leukemic BCR-ABL mutant cells, possibly allowing ponatinib resistant clones to be overcome. Disclosures: Ohyashiki: Novartis: Honoraria, Research Funding.

Description: Abstract 2136 Imatinib has shown clinical efficacy against Philadelphia chromosome (Ph) positive leukemia cells and it is now the standard care for initial therapy. However, recent studies reported imatinib are not effective in quiescent primitive chronic myeloid leukemia (CML) stem cells. Moreover, many Ph-positive leukemia patients develop resistance or fail to respond to imatinib by mutation in the ABL kinase domain in clinically. These results indicated that alternative combination therapy such as BCR-ABL targeting tyrosine kinase inhibitors (TKIs) and nontoxic agents are required to cure the Ph positive leukemia patients. Hedgehog (Hh)- Glioma-associated oncogene homolog (Gli) signaling regulates self-renewal of stem cells and implicates in a large number of human cancers. One of the Hh inhibitor, GDC-0449 is a potent small molecule inhibitor of Hedgehog-Gli pathway. It has been reported GDC-0449 showed high target specificity and demonstrated antiproliferative activity against tumors and it is now in clinical trial. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Hedgehog-Gli inhibitor, GDC-0449 may help prevent CML relapse and these approaches may be expected to improve the outcomes of Ph-positive leukemia patients. In this study, we investigated the GDC-0449 efficacy by using the BCR-ABL positive cell lines, OM9;22, K562 and primary samples when leukemic cells were protected by the feeder cell line, S9 cells. We examined a comprehensive drug combination experiment using GDC-0449 and dual Src/ABL tyrosine kinase inhibitor, dasatinib. Gli proteins (Gli1, Gli2 and Gli3) were existed in Ph-positive cell lines. We found the cell numbers of OM9;22 were significantly increased with the feeder cell line, S9 cells compared to without S9 cells. The treatment of dasatinib exhibits cell growth inhibition partially against OM9;22 cells in the presence of feeder cell line, S9 cells. Caspase-3 activity by 100 nM dasatinib treatment was also reduced in the presence of S9 cells. 72 h of combined treatment of Ph-positive leukemia cells with 10 μM of GDC-0449 and 100 nM of dasatinib in the presence of feeder cell line, caused significantly more cytotoxicity than each drug alone. We next investigated the efficacy and intracellular signaling of GDC-0449. The treatment of GDC-0449 exhibits cell growth inhibition and induced apoptosis against OM9;22 cells in a dose and time dependent manner. Expression of Gli1 and Gli2 proteins were reduced after GDC-0449 treatment. 10 μM of GDC-0449 also inhibited the growth of Ph-positive primary samples by colony assay. Another Hh inhibitor, SANT-2 also exhibits cell growth inhibition against OM9;22 cells in a dose dependent manner. Data from this study suggested that administration of the Hh inhibitor, GDC-0449 may be a powerful strategy against Ph-positive leukemia cells and enhance cytotoxic effects of dasatinib in the presence of feeder cell. Disclosures: Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.

Description: Abstract 4460 The clinical use of imatinib, a specific BCR-ABL tyrosine kinase inhibitor (TKI) is effective in inducing a complete hematological and cytogenetic remission in a high percentage of chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph) positive acute lymphoblastic leukemia (ALL) patients. However, imatinib does not efficiently kill leukemic stem cells and is limited by the emergence of resistance due to the point mutations in the BCR-ABL kinase domain. Histone acetyltransferases (HAT) and histone deacetylases (HDAC) control the acetylation of histones and intracellular proteins, and regulate the transcription and function of the proteins. HDAC inhibitor is a structurally diverse class of targeted anti-cancer agent. One of the pan-HDAC inhibitor, vorinostat (suberoylanilide hydroxamic acid: SAHA) is a small-molecule inhibitor of most human class I and class II HDAC, and is reported the efficacy of malignant cells including lymphomas and myeloid malignancies.Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitor and an HDAC inhibitor, vorinostat may help prevent CML relapse due to BCR-ABL point mutation and may improve their long-term outcome. In this study, we investigated the efficacy of vorinostat by using the Ph-positive leukemia cell line, K562 and Ba/F3 BCR-ABL cell in a random mutagenesis study for BCR-ABL mutation. We first performed a comprehensive drug combination experiment using vorinostat and BCR-ABL tyrosine kinase inhibitor, imatinib or nilotinib. The treatment of imatinib or nilotinib exhibits cell growth inhibition partially against Ba/F3 BCR-ABL cell in a random mutagenesis. We also found the BCR-ABL point mutation such as T315I or M344V after 2 weeks nilotinib treatment by direct sequence analysis. We show that vorinostat potently induced cell growth inhibition of K562 and Ba/F3 BCR-ABL cells in a random mutagenesis in a dose dependent manner. Combined treatment of Ba/F3 BCR-ABL cell in a random mutagenesis with vorinostat and nilotinib or imatinib caused significantly more cytotoxicity than each drug alone by colony assay. We investigated the intracellular signaling of vorinostat. Phosphorylation of BCR-ABL, Crk-L were reduced after vorinostat treatment for 24 hours in a dose dependent manner. Caspase 3 and poly (ADP-ribose) polymerase (PARP) activation were increased after vorinostat treatment. Vorinostat potently enhanced cell growth inhibition of Ba/F3 BCR-ABL point mutants (G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T and H396P) compared with Ba/F3 expressing Wt BCR-ABL cells. The protein level of BCR-ABL was reduced after vorinostat treatment. BCR-ABL degradations in BCR-ABL mutant cells were significantly enhanced compared with Ba/F3 Wt BCR-ABL cells. Although long term culture of Ba/F3 BCR-ABL cell in a random mutagenesis with 2μ M vorinostat significantly decreased cell growth, the cells were increased after removal of vorinostat. We found these cells were wild type BCR-ABL by direct sequence analysis. Data from this study suggested that administration of the vorinostat may mediate its effects on BCR-ABL positive cells included BCR-ABL point mutation and enhance cytotoxic effects of nilotinib or imatinib in BCR-ABL mutant cells, and provide information of potential therapeutic relevance. Disclosures: Ohyashiki: Nippon Shinyaku Co., Ltd.: Research Funding.

Description: Abstract 3417 Background: Now, imatinib has been established as a first line therapy for the patients with chronic myelogenous leukemia (CML) in chronic phase (CP), and a standard initial dose is 400 mg daily. Although less than 300 mg showed unsatisfactory results, 300 mg or more appears effective in some patients intolerant to standard dose. However, large studies have not yet explored the response to lower dose of imatinib. In this CML202 study, although initial dose of imatinib was scheduled to be 400 mg, many patients actually received reduced dose mainly due to adverse events. However, overall efficacy and outcomes had been comparable to other studies. We performed subgroup analysis regarding long-term survivals according to the mean daily dose during the first 6 months, 12 months, and 24 months, respectively. We also measured imatinib plasma trough concentration (Cmin) in patients receiving imatinib at a dose of 300 mg or 400 mg, and compared efficacy and survivals between them. Methods: The prospective multicenter study of imatinib therapy in Japanese patients with newly diagnosed CML-CP was conducted in Japan Adult Leukemia Study Group (JALSG CML202 study). The objectives of this study were to determine the efficacy, safety and long-term outcomes of imatinib therapy in patients with newly diagnosed CML-CP. Primary end point of imatinib therapy was overall survival (OS). Initial daily dose of imatinib was 400 mg. The plasma concentration of imatinib was measured using liquid chromatography-tandem mass spectrometry. Results: 488 patients were enrolled between 2002 and 2006, and data at a median follow-up of 66 months were analyzed. The cumulative best response rates of major cytogenetic response (MCyR), complete cytogenetic response (CCyR) and major molecular response (MMR) were 99%, 98% and 81%, respectively. At 66 months, the estimated rate of overall survival (OS) was 93% (95% CI, 90 to 96), and that of survival without progression to accelerated phase (AP) or blast crisis (BC) was 97% (95% CI, 96 to 99). In the landmark analysis at 12 months or 18 months according to the cytogenetic response or the molecular response, patients who had CCyR or MMR showed significantly better event free survival (EFS) rates than those who had not, respectively. Mean daily doses of imatinib administered during the first 24 months were 400 mg or more (400 mg group) in 294 patients, less than 400mg and 300mg or more (300 mg group) in 108 patients, and less than 300 mg (200 mg group) in 86 patients. The efficacy and outcomes at 66 months were evaluated according to the mean daily dose (400, 300, 200 mg group). The estimated rates of OS were 98%, 92% (P=0.09), and 74% (P

Description: Hematopoietic stem cells and leukemic stem cells share common features, including self-renewal, the capacity to differentiate, resistance to apoptosis, and limitless proliferative potential. Despite these similarities, several stemness factors, such as Hedgehog, Wnt, Notch, and Dopamine Receptor show differential activation in normal versus leukemia stem cells. Using a high-throughput discovery platform that reveals differences between neoplastic and normal human pluripotent stem cells, the norvel dopamine receptor antagonist, thioridazine, was identified from libraries of known compounds that induce differentiation to overcome neoplastic self-renewal (Cell 149, 1284, 2012). In the present study, we investigated the molecular mechanisms by which dompamine receptor antagonist, thioridizine, regulates the self-renewal of primary BCR-ABL1 positive leukemia cells in vivo. To identify the leukemia-propagating cell fraction of BCR-ABL1-positive leukemia, we serially transplanted human leukemia cells from patients with chronic myeloid leukemia blast crisis (n=1; T315I BCR-ABL1) or ponatinib-resistant Ph-positive acute lymphoblastic leukemia (n=2, Y253H/E255K/T315I BCR-ABL1 and T315I BCR-ABL1) into NOD/SCID/IL-2γc-/- mice. The cell fractions with CD34+CD38- CD19+and CD34+CD38+CD19+ could self-renew and transfer the leukemia in NOD/SCID mice. To investigate the effects of the domamine receptor antagonist on self-renewal and the relevance as a therapeutic target in ABL-tyrosine kinase-resistant BCR-ABL1 positive leukemia, we examined the activity of thioridazine against CD34+CD38-CD19+, CD34+CD38+CD19+ fractions transferred NOD/SCID mice in vivo. NOD/SCID mice were injected intravenously with BCR-ABL1 positive cells then treated with thioridazine (20 mg/kg; p.o.) for 28 days. All mice demonstrated the engraftment of leukemia by flow cytometry. However, the treatment with thioridazine reduced the population of CD34+CD38- positive cells. We isolated human CD45+ cells from the spleen of mice from each treatment group and injected equivalent numbers of leukemia cells into secondary recipients. Following 30 days, all mice received BCR-ABL1 cells from vehicle treated mice engrafted with leukemia. In contrast, leukemia engraftment was not detected in recipient mice (n=6) from thioridazine-treated donors. These results demonstrate the persistent effects of domapine receptor signaling inhibition on long term self-renewing BCR-ABL1-positive leukemia cells. We further examined the effects of dompamine receptor pathway modulation on in vitro clonogenic growth. CD34+CD38-CD19+ cells from T315I BCR-ABL1 (n=2) and WT-BCR-ABL1 (n=1) cells were treated with 5 μM of thiorizaxine for 72 hrs, washed free of drugs, and plated in quadruplicate in methylcellulose. At 14 days, colonies were counted as initial plating. The representative plate was then washed and cells were re-suspended and re-plated. After an additional 14 days, colonies were counted as secondary re-plating. Clonogenic recovery of untreated cells was normalized to 100% and plating results from all treatment groups were expressed as % control. Dopamine receptor pathway inhibition by thioridazine had only minimum effects on colony formation after initial plating over control cells. However, upon serial re-plating, secondary colony formations were significantly inhibited by thioridazine (p

Description: Multiple myeloma (MM) is one of the common hematological malignancies and is a uniformly fatal disorder of B cells characterized by accumulation of abnormal plasma cells in the bone marrow. Proteasome inhibitor, bortezomib, and immunomodulatory drugs such as thalidomide and lenalidomide play important roles in the treatment of MM patients. Although novel agents including, e.g. bortezomib, have significantly improved the response and survival of patients with MM, a large number of patients eventually have relapsed. For the patients who relapse after treatment with novel agents, the prognosis is still poor. Thus circumstanced, alternative strategies are required for continued disease control. Phosphoinositide 3-kinases (PI3Ks) are a family of proteins involved in the regulator of cell growth, metabolism and proliferation. PI3K signaling pathway also plays a critical regulatory role in MM pathology, including survival, cellular proliferation, migration and angiogenesis. Therefore, PI3K signaling pathway may present attractive targets for MM treatment. Copanlisib also known as BAY80-6946 is a potent and highly selective reversible PI3K inhibitor. Copnalisib is currently investigated in a pivotal phase 2 clinical trial against hematological malignancy such as malignant lymphoma. We hypothesized that treatment with PI3K inhibitor and proteasome inhibitors together would result in enhanced therapeutic activity in MM cells. In this study, we investigated the efficacy of copanlisib by using the MM cell lines, RPMI8226, MM1.S and MM1.R and primary sample. 72 h treatment of copanlisib exhibits cell growth inhibition of MM cell lines in a dose dependent manner. The treatment of proteasome inhibitors, bortezomib and carfilzomib exhibits cell growth inhibition partially against RPMI8226 cells in the presence of feeder cell line, HS-5. We examined the intracellular signaling in the presence of HS-5. Phosphorylation of Akt and activation of caspase 3 and poly (ADP-ribose) polymerase (PARP) was partially reduced by carfilzomib or bortezomib in the presence of HS-5. We found that the treatment of copanlisib abrogated the protective effects of HS-5 in RPMI8226 cells. We examined the intracellular signaling after treatment of copanlisib. Activity of caspase 3 and poly (ADP-ribose) polymerase (PARP) was increased after copnlisib treatment in a dose dependent manner. Because PI3K signaling pathway regulates MM cell migration, we next evaluated the chemotactic response of MM cells to stromal cell-derived factor 1α (SDF-1α). We found that 4 h treatment of SDF-1α significantly induced the migration of MM cells compared to control medium. Treatment of copanlisib inhibited SDF-1α-stimulated chemotaxis in a dose dependent manner. We found that phosphorylation of Akt was reduced after copanlisib treatment suggesting that intracellular PI3K signaling pathway may play the important role in SDF-1α induced chemotaxis of MM cells. We investigated the copanlisib activity against MM cells. Combined treatment of MM cells with proteasome inhibitor, carfilzomib or bortezomib, and copanlisib caused significantly more cytotoxicity than each drugs alone. Phosphorylation of Akt was reduced and cleaved PARP was increased after copanlisib with or without proteasome inhibitor. We also found that copanlisib which was combinaed with carfilzomib or borteomib exhibited cell growth inhibition against MM primary sample. Data from this study suggested that administration of the PI3K inhibitor, copanlisib may be a powerful strategy against stroma-associated drug resistance of MM cells and enhance cytotoxic effects of proteasome inhibitors in those residual MM cells. Disclosures No relevant conflicts of interest to declare.