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Abstract 2651 Poster Board II-627 The oncoprotein PIM1 is a cytoplasmic serine/threonine kinase, which is overexpressed and implicated in the pathogenesis of hematologic malignancies. PIM1 activity promotes cell survival and proliferation, and is a downstream mediator of cytokine receptor signaling involving the JAK/STATs. PIM1 phosphorylates and inactivates Bad, which enhances the anti-apoptotic functions of Bcl-2. PIM1 also mediates the phosphorylation and inactivation of cell cycle inhibitors p21 and p27, thereby promoting progression through G1 and G2/M phases of the cell cycle. PIM1 phosphorylates serine 10 on histone H3, and has been shown to phosphorylate and stabilize Myc, thereby collaborating in Myc-induced transformation. Further, PIM1 is known to phosphorylate 4EBP1 on the same residues (threonine 37 and 46) as mTOR kinase. It can also phosphorylate eIF4B, thereby maintaining high levels of protein translation. PIM1 is known to be chaperoned as a heat shock protein 90 client protein. In the present studies, we demonstrate that treatment with the PIM kinase inhibitor SGI-1776 (SuperGen Inc.) dose-dependently (500 nM to 2.0 μM) depleted p-BAD (Ser112), p-p70S6K, p-4EBP1, p-Ser10 Histone H3 and c-Myc levels, while inducing p27 and p21 expression, in the cultured human CML-BC K562 and erythroleukemia HEL92.1.7 (with homozygous JAK2-V617F mutation). Treatment with SGI-1776 induced apoptosis of K562 and HEL92.1.7 (HEL) cells as well as Ba/F3-hEpoR-JAK2-V617F but not Ba/F3-hEpoR cells. Similar effects of SGI-1776 were observed in primary CML-BC and MPN (myeloproliferative neoplasm) cells. Treatment with the non-geldanamycin analogue hsp90 inhibitor AUY922 (Novartis Pharma) or with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharma) disrupted the chaperone association of PIM1 with hsp90 and increased binding to hsp70, thereby promoting the proteasomal degradation of PIM1 in K562 and HEL cells. Treatment with PS (20–100 nM) also depleted PIM1 mRNA as measured by qPCR. Co-treatment with SGI-1776 (0.5 to 2.0 μM) and clinically achievable concentrations of AUY922 (e.g., 25 nM) caused greater depletion of pBAD (Ser112), pp70S6K, p4EBP1, cyclin B1, c-Raf, c-Myc, and AKT and synergistically induced apoptosis of K562 and HEL92.1.7 cells (combination indices < 1.0). As compared to treatment with either agent alone, co-treatment with SGI-1776 and AUY922 or PS also exerted greater cytotoxicity against primary CML-BC and MF-MPN cells. Importantly, co-treatment with PS significantly enhanced SGI-1776-induced apoptosis of purified primary CD34+CD38-Lin- hematopoietic stem cells expressing JAK2-V617F derived from a patient with primary myelofibrosis. Taken together, these pre-clinical findings indicate that simultaneously depleting PIM kinase levels by AUY922 or PS and inhibiting PIM1 kinase activity with SGI-1776 induces synergistic apoptosis against cultured and primary CML-BC and MPN cells. Additionally, our findings suggest that co-treatment with PS and SGI-1776 may be an effective treatment strategy against hematopoietic stem cells expressing JAK2-V617F in patients with MF-MPN. Disclosures: Atajada: Novartis: Employment. Quadt:Novartis: Employment. Bhalla:Merck: Honoraria; Novartis: Honoraria, Research Funding.

Multiple myeloma still is an incurable disease in the majority of patients and novel treatment strategies are urgently needed. Recently, heat shock protein (HSP) 90 has been identified as a novel therapeutical target in multiple myeloma. Inhibition of HSP 90, e.g. with NVP-AUY922, has shown in vitro activity against multiple myeloma by destabilization of pro-survival proteins like Akt, and clinical studies with HSP 90 inhibitors are ongoing. Little is known to date about possible synergistic drug interactions for effective combination treatment with HSP 90 inhibitors. Histone deacetylase inhibitors (HDAI) like NVP-LBH589 or SAHA promote hyperacetylation of histones, transcription factors and signaling molecules, leading to modified gene transcription and signaling pathways, finally resulting in cell death in multiple myeloma. Since HDAIs also promote HSP 90 hyperacetylation, we postulated synergistic effects between HDAIs and the HSP 90 inhibitor NVP-AUY922. Multiple myeloma cell lines (OPM-2, MM1.S, RPMI-8226) and primary cells were exposed to NVP-AUY922 in combination with one of the HDAIs NVP-LBH589 or SAHA. Cells were treated in simultaneous or sequential incubation patterns, respectively. Viability of cells was quantified by MTT assay and apoptosis was determined by annexin-V assay. Testing for synergistic effects of combination experiments was performed using the median effect method by Chou and Talalay. Relative HSP90 client protein concentration and phosphorylation were determined by intracellular staining and phospho-FACS flow cytometry. After 72 hours of incubation, IC50 viability values for SAHA and NVP-LBH589 were determined at 0.8 μM and 6.6 nM for OPM-2 and 0.6 μM and 5.5 nM for MM1.S. Incubation for 72 hours with NVP-AUY922 resulted in IC50 of 31 nM and 15 nM for OPM-2 and MM1.S cell lines, respectively, whereas RPMI-8226, even at concentrations >100 nM showed a maximum inhibition of 50 % in comparison to controls. Incubation for 24 hours with SAHA and addition of NVP-AUY922 for another 48 hours for combined exposure resulted in synergistic cytotoxic effects with a combination index (CI) < 0.7, whereas simultaneous incubation for 72 hours or 24 hours pre-incubation with NVP-AUY922 and subsequent 48 hours combined treatment did not show synergistic effects (CI ≥ 1.0) in OPM-2 cell line. Synergistic effects with CI < 1.0 were also achieved in MM1.S and RPMI-8226 when incubated with SAHA or NVP-LBH589 and subsequent combined treatment with NVP-AUY922. Interestingly, in the less responsive cell line RPMI-8226, SAHA doses well below IC50 resulted in a strong synergism with NVP-AUY922 (CI < 0.5). Apoptosis analysis showed an overproportional increase in annexin-V-positive cells for combination treatment (41% specific apoptosis) in comparison to single agent exposure in OPM-2 (12% and 22% for 50 nM NVP-AUY922 and 0.8 μM SAHA, respectively). Intracellular phospho-FACS analysis showed an increase in HSP 70 and a decrease in Akt, p-Akt and p-ERK concentration after 24 hours incubation with NVP-AUY922. In conclusion, this is the first report on synergistic effects of NVP-AUY922 and HDAIs in multiple myeloma. Additionally, we were able to define the most effective sequence of drug administration. These data provide a framework for future clinical studies with the combination of both inhibitors in patients with multiple myeloma.

The mutant JAK2-V617F is present and constitutively active in myeloproferative disorders (MPD), including polycythemia vera (PV), essential thrombocytosis and primary myelofibrosis (MF). JAK2-V617F activates the signal transduction through the STAT, RAS/MAPK and PI3K/AKT pathways, conferring proliferative and survival advantage in the MPD hematopoietic progenitor cells (HPCs). We have previously reported that the panhistone deacetylase (HDAC) inhibitors e.g., panobinostat (Novartis Pharmaceuticals Inc), induces growth arrest and apoptosis of neoplastic HPCs. Panobinostat (PS), by inhibiting HDAC6, induces hyperacetylation of heat shock protein (hsp) 90, which attenuates the ATP binding and chaperone function of hsp90. This is associated with polyubiquitylation and degradation of hsp90 client protein signaling kinases, e.g, FLT-3, Bcr-Abl, AKT and c-RAF. Additionally, the mutant versions of signaling oncoprotein kinases, e.g., FLT-3, Bcr-Abl, B-RAF and c-KIT, may be even more dependent on hsp90 chaperone function for maintaining their proper folding and active conformation. This makes the mutant oncoprotein kinase even more susceptible to misfolding and proteasomal degradation upon inhibition of hsp90 function. In the present studies we determined the effects of PS or the non-geldanamycin analogue hsp90 inhibitor (AUY922, Novartis) and/or the JAK-2 inhibitor TG101209 (TG) (TargeGen, San Diego, CA) in the cultured (human erythroleukemia HEL and mouse BaF3 cells expressing JAK2-V617F) and primary human MPD cells (from two patients with PV and MF. Treatment with PS (10 to 30 nM) dose-dependently increased % of cells in G1 and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of HEL and BaF3/JAK2-V617F cells. This was associated with attenuation in the levels of JAK2-V617F, p-JAK2, p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310), GATA1 and Bcl-xL in HEL cells. PS induced lysine acetylation of hsp90 but not of JAK2. Similar effects were observed, following treatment with AUY922 (AUY). Both PS and AUY induced significantly less apoptosis of BaF3/hEpoR versus BaF3/JAK2-V617F cells. PS and AUY also reduced the chaperone association of JAK2-V617F with hsp90. Treatment with TG (0.2 to 2.0 uM) also dose-dependently attenuated the levels of p-STAT3, p-STAT5, p-AKT (serine 473), AKT, p-ERK1/2, p-GATA1 (serine 310) and Bcl-xL, which was associated with apoptosis of HEL and BaF3/JAK2-V617F cells. Co-treatment with PS and TG induced more apoptosis of the cultured and primary MPD cells than treatment with either agent alone. AUY also increased apoptosis of HEL cells induced by TG. Co-treatment with PS and AUY synergistically induced apoptosis of HEL cells (combination indices < 1.0). Co-treatment with 20 nM of PS or 15 nM of AUY augmented TG mediated attenuation of p-STAT3, p-STAT5 and Bcl-xL levels in HEL and BaF3/JAK2-V617F cells. These findings demonstrate for the first time that pan-HDAC inhibitor PS and hsp90 inhibitor AUY attenuate the levels and signaling of JAK2-V617F and enhance the anti-JAK2-V617F activity of TG in human MPD cells. Our findings also support the rationale to determine the in vivo activity of PS or AUY in combination with JAK2 inhibitors against human MPD.

Context: The molecular chaperone hsp90 is a major anti-cancer therapeutic target because it regulates the function of proteins with pivotal roles in tumor cell proliferation, survival and drug resistance, including mutated/chimeric oncoproteins or oncogenic kinases/receptors. Our preclinical studies on the ansamycin hsp90 inhibitor tanespimycin (17-AAG) provided the rationale for clinical trials, either alone or in combination with the proteasome inhibitor bortezomib, for treatment of relapsed/refractory MM. In this study, we report preclinical studies of the new, non-ansamycin, hsp90 inhibitor NVP-AUY922. Methods/Results: We tested 36 human MM cell lines and observed with MTT colorimetric survival assays potent time- and dose-dependent anti-MM activity of NVP-AUY922. IC50 values were Conclusion: The new hsp90 inhibitor NVP-AUY922 has potent in vitro activity against MM cells resistant to conventional therapeutics, with selectivity for malignant compared to normal cells. Ongoing in vivo experiments and studies to identify biomarkers of pronounced sensitivity to NVP-AUY922 will help provide a framework for potential clinical trials of NVP-AUY922 in MM and other neoplasias.

We have recently shown that Hsp90 is overexpressed in multiple myeloma (MM) and critically contributes to tumor cell survival. Pharmacologic blockade of Hsp90 has consistently been shown to induce MM cell death. However, most data have been obtained with MM cell lines whereas knowledge about the molecular effects of pharmacologic Hsp90 blockade in primary tumor cells is lacking. Furthermore, these investigations have so far exclusively relied on geldanamycin derivatives. Here, we analyzed the anti-tumor effects of a novel diarylisoxazole-based Hsp90 inhibitor (NVP-AUY922) in a large set of primary MM samples and in MM cell lines. The majority of cell lines (n = 8), as well as most primary samples (n = 20), displayed profound apoptotic responses and steep dose-effect curves with EC50 values in the range of 5–15 nM and EC90 values between 8 and 25 nM. This effect was not attenuated in coculture with cells from the bone marrow microenvironment. Some cell lines and about a quarter of primary MM samples displayed greater resilience to drug treatment, with EC50 values but not EC90 values reached at concentrations up to 50 nM. Sensitivity of MM cells to the Hsp90 inhibitor was not correlated with TP53 mutation or Hsp70 induction levels. Western analyses of MM cell lines and flow cytometric analyses of antibody-stained Hsp90 client proteins in primary tumor cells showed that NVP-AUY922-treatment entailed molecular effects and pharmacodynamic properties consistent with abrogation of Hsp90 function. Consequently, downregulation of multiple signaling and survival pathways was detectable through, for example, decreases in the phosphorylated (activated) forms of extracellular signal-regulated kinase (ERK) 1 and 2, signal transducer and activator of transcription (STAT) 3 and glycogen synthase kinase-3beta. All samples treated displayed strong upregulation of Hsp70. Importantly, peripheral blood mononuclear cells as well as primary bone marrow stromal cells were much less affected by high (50–100 nM) concentrations of NVP-AUY922, showing that a therapeutic window might be established for the treatment of multiple myeloma. Taken together, NVP-AUY922 could be a promising new drug for the treatment of a majority of myeloma patients.