ALBERT

Beschreibung: Introduction: CSF1R (M-CSFR) is a receptor tyrosine kinase that, upon binding with CSF1 ligand (M-CSF), activates downstream survival, proliferation, and differentiation pathways in myeloid lineage cells. Expression and activity of CSF1R has been shown to be important for sustaining tumor-associated macrophages (TAMs) in a variety of solid tumor types, and these TAMs have been shown to provide a supportive microenvironment that maintains tumor cell viability and growth. Monocyte and macrophage lineage cells have also been implicated in providing a supportive microenvironment in certain hematologic malignancies, such as nurse-like cells in CLL, but have not been extensively explored in AML. Using an ex vivo functional screening platform applied directly to primary specimens from AML patients, we have identified recurrent sensitivity to CSF1R inhibition or silencing due to a depletion of CSF1R-expressing monocytic-lineage cells in the AML microenvironment. Methods: To identify new therapies for AML patients, our lab has screened hundreds of AML patient specimens against panels of targeted small-molecule inhibitors or siRNA and evaluated the impact on cell viability. These panels have included two small-molecule inhibitors with exquisite specificity for CSF1R (GW-2580 and ARRY-382). We have also used a panel of recombinant growth factors and cytokines, including CSF1, to measure their impact on stimulating AML cell growth. Results: We found that 20-30% of AML specimens showed sensitivity to inhibition or silencing of CSF1R, with good concordance between CSF1R siRNA, GW-2580, and ARRY-382. Exhaustive analysis of the clinical, demographic, and genetic features of these patients failed to reveal correlation between CSF1R and any prominent disease subsets. Samples sensitive to CSF1R silencing or inhibition are significantly more likely than non-sensitive samples to respond to CSF1 growth stimulation, indicating a ligand-dependent mechanism of CSF1R signaling. Additionally, we examined CSF1R and CSF1 expression in discrete hematopoietic cell populations by flow cytometry in our specimens, both with and without CSF1R inhibition. Interestingly, we found that CSF1R was predominantly expressed a subpopulation of CD14-expressing monocytes, not tumor cells, and this population disappeared after exposure to the CSF1R inhibitor. Moreover, we identified a correlation between sensitivity to CSF1R inhibition and the presence of this CD14/CSF1R-expressing cell population. Conclusions: These results suggest that CD14/CSF1R-expressing cells support AML blasts through a ligand-dependent mechanism, and depleting these cells eliminates the supportive microenvironment, resulting in leukemia cell death. Therefore, we propose using CSF1R inhibitors, such as ARRY-382, as a promising new line of therapy to target AML by disrupting the tumor microenvironment. Disclosures Agarwal: CTI BioPharma: Research Funding. Lee:Array Biopharma: Employment. Chantry:Array Biopharma: Employment. Druker:Sage Bionetworks: Research Funding; Millipore: Patents & Royalties; Molecular MD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; McGraw Hill: Patents & Royalties; CTI Biosciences, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; AstraZeneca: Consultancy; Oregon Health and Science University: Patents & Royalties; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Oncotide Pharmaceuticals: Research Funding; Novartis Pharamceuticals: Research Funding; ARIAD: Research Funding; Aptose Therapeutics Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tyner:Incyte: Research Funding; Janssen Pharmaceuticals: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Array Biopharma: Research Funding.

Beschreibung: We report here the biological effects of inhibiting Pim-1, -2 and -3 serine/threonine protein kinases in cells and in vivo using a novel, potent and selective small molecule inhibitor directed against these kinases. In vitro enzyme assays revealed potent inhibition of all three Pim kinase isoforms while maintaining selectivity against more than 220 other protein kinases. In cells, compound treatment produced biological effects consistent with Pim inhibition, including a reduction in BAD protein phosphorylation. Moreover, this biological activity corresponds well with the compound’s anti-proliferative and pro-apoptotic activity in the IL-3 dependent mouse pro-B cell line Ba/F3 and in factor-indpendent Ba/F3 cell lines dependent upon the expression of BCR-Abl or TEL-JAK2 for growth and survival. Importantly, compared to the BCR-Abl driven Ba/F3 cell line, this compound was equally effective at blocking the growth of an Imatinib-resistant BCR-Abl[ T315I]-driven Ba/F3 cell line. Finally, this compound possesses pharmacokinetic properties that predict the potential for in vivo activity. Therefore, we assessed the effect of Pim kinase inhibition on tumor growth in a mouse subcutaneous tumor xenograft model employing a cell line driven by the TEL-JAK2 oncogene. Results demonstrated that the compound significantly inhibited tumor growth in a dose-dependent manner. In terms of potential clinical utility, these data show that Pim kinase inhibition could be an effective therapeutic strategy for numerous hematologic malignancies including chronic and acute myelogenous leukemias, particularly in those patients that have become resistant to existing therapies such as Imatinib.

Beschreibung: Multiple myeloma (MM) is characterized by the expansion of malignant plasma cells within the bone marrow. Their growth, survival, and migration are mediated in part via cytokines. Interleukin 6 (IL-6) is necessary for sustaining the in vitro growth of many MM cell lines and enhancing the proliferation of explanted human myeloma cells. The mitogen-activated protein kinase family member, p38, is activated by cytokines and growth factors and plays a significant role in inflammatory diseases. However, its role in the pathogenesis of multiple myeloma is poorly understood. Specific p38 inhibitors inhibit paracrine MM cell growth which is associated with IL-6 and VEGF secretion from bone marrow stromal cells (BMSCs). Furthermore, p38 inhibition blocks TNF-alpha-induced IL-6 secretion in BMSCs, thereby further inhibiting MM cell growth and survival. Although these data suggest an important role for p38 in MM, the direct effects of p38 inhibiton on MM has not been extensively explored. Therefore, we investigated the effects of p38 inhibition on in vitro and in vivo IL-6 production and MM cell growth in vivo after lipopolysaccaride (LPS) stimulation. LPS has been shown to induce various cytokines, including TNF-alpha and IL-6, via the p38 pathway. ARRY-797, an orally bioavailable, small molecule inhibitor of p38 directly inhibited LPS-induced IL-6 production from RPMI-8226 (IC50 = 100 pM) in vitro. In SCID-beige mice, LPS (3 μg/kg) induced IL-6 (7897 ± 827 pg/mL) and TNF-alpha (1922 ± 282 pg/mL) after 2 hours and these cytokines were inhibited by oral administration of ARRY-797 (30 mg/kg) by 91% and 95%, respectively. In MM xenograft models, ARRY-797 (30 mg/kg, BID, PO) inhibited RPMI 8226 tumor growth by 72% as a single agent and by 56% when LPS was administered to stimulate growth in vivo. In addition, ARRY-797 inhibited LPS-induced phosphorylation of p38 in RPMI-8226 xenografts. Together, these data support a role for p38 in IL-6-mediated growth of multiple myelomas. To our knowledge, ARRY-797 is the first small molecule p38 inhibitor to demonstrate single agent activity in a MM xenograft model and it has been advanced into preclinical development.

Beschreibung: Introduction: Monocyte/macrophage lineage cells have been reported to provide a supportive signal in a variety of neoplastic settings. Tumor-associated macrophages (TAMs) have been shown to provide microenvironmental support that maintains tumor cell viability and growth for a variety of solid tumor types, and these TAMs have been shown to depend on the receptor tyrosine kinase, CSF1R (M-CSFR). Monocyte/macrophage lineage cells have also been implicated in the microenvironment of CLL and are termed nurse-like cells in this setting. However, the role of CSF1R in CLL including potential maintenance of these nurse-like cells has not been explored. Using an ex vivo, functional screening platform applied directly to primary specimens from CLL patients, we have identified recurrent sensitivity to CSF1R inhibitors as well as decreased viability after depletion of CSF1R-expressing monocytes, thereby depriving the CLL cells of an important, microenvironmental growth/survival signal. We also have seen synergistic anti-tumor activity when combining CSF1R inhibitors with idelaisib and ibrutinib which inhibit b-cell receptor (BCR) activated pathways. Methods: We evaluated the impact on cell viability of hundreds of CLL patient specimens against panels of targeted small molecule inhibitors. These panels include two small-molecules with exquisite specificity for CSF1R (GW-2580; ARRY-382). In addition, we evaluated the impact of antibody depletion of monocytes (CD14-depletion) on ex vivo CLL cell viability as well as the effect of monocyte cell depletion on response to CSF1R inhibitors. We also evaluated the combination of GW-2580 or ARRY-382 with idelalisib (PI3kδ inhibitor) and ibrutinib (BTK inhibitor). Results: We found that 20-30% of CLL specimens showed sensitivity to inhibition of CSF1R with good concordance between GW-2580 and ARRY-382. Analysis of clinical and demographic features of these patients failed to reveal correlation of CSF1R with any prominent disease subsets. Flow cytometry analysis revealed that CSF1R was not expressed on CLL cells but only on a subpopulation of CD14-expressing monocytes. Depletion of these monocytes with CD14 antibody had little to no impact on samples not exhibiting ex vivo sensitivity to CSF1R inhibitors, however, samples showing strong sensitivity to CSF1R inhibitors were also quite sensitive to depletion of this CD14-positive monocyte population. After CD14 depletion, the remaining CLL cells showed no further sensitivity to CSF1R inhibitors. Finally, when combined with ibrutinib or idelalisib synergy was seen. Conclusions: These results show that CSF1R is a potential therapeutic target in CLL and suggest that CLL supporting monocytes (nurse-like cells) express CSF1R and depend on CSF1R for viability in a similar manner as TAMs depend on CSF1R in a variety of solid tumor settings. As such, CSF1R inhibitors may deprive CLL cells of this supportive microenvironmental signal resulting in CLL cell death. Therefore, we propose that CSF1R inhibitors, such as ARRY-382, possibly in combination with ibrutinib, idelalisib, or other approved agents, may be a promising new line of therapy to target CLL cells by impacting the tumor microenvironment. Disclosures Spurgeon: Genentech: Honoraria; Acerta Pharma: Research Funding; Bristol Meyers Squibb: Research Funding; Gilead sciences: Honoraria, Research Funding; Janssen: Research Funding; Pharmacyclics: Honoraria. Tyner:Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding; Aptose Biosciences: Research Funding; Constellation Pharmaceuticals: Research Funding; Array Biopharma: Research Funding. Agarwal:CTI BioPharma: Research Funding. Lee:Array Biopharma: Employment. Chantry:Array Biopharma: Employment. Druker:Fred Hutchinson Cancer Research Center: Research Funding; Bristol-Myers Squibb: Research Funding; Henry Stewart Talks: Patents & Royalties; Millipore: Patents & Royalties; Sage Bionetworks: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Consultancy; Novartis Pharmaceuticals: Research Funding; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotide Pharmaceuticals: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; ARIAD: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Beschreibung: Abstract 2858 Poster Board II-834 Introduction: The allosteric kinesin spindle protein (KSP) inhibitor ARRY-520 has potent antitumor activity as a single agent in xenograft models of multiple myeloma, with complete response rates as high as 100% in some models (Eur J Cancer Supp 6(12): 447, 2008). These observations led to a phase 1 clinical trial (currently in progress) in patients with advanced / refractory multiple myeloma who have failed treatment with both bortezomib and an imid. In further preclinical work, we report the activity of ARRY-520 in combination with standard agents used for the treatment of multiple myeloma, bortezomib and revlimid. Methods: RPMI8226, JJN3 and H929 multiple myeloma tumor xenografts were grown in female SCID-beige mice by implanting 0.5 –2 × 107 cells subcutaneously in the right flank. When tumors reached a volume of ∼ 150 – 250 mm3, mice were randomized into treatment groups based on tumor volume. Compounds were administered on schedules similar to those used in the clinic: bortezomib twice a week for two weeks, revlimid QD, and a single course of ARRY-520 on d1,2. ARRY-520 was formulated in normal saline and administered IP. Bortezomib was formulated following the supplier's instructions and also administered IP. Revlimid was formulated in 1% CMC / 0.5% Tween-80 and administered PO. Animal weights and tumor volumes were measured two or three times a week during the course of the experiments. Results: In mice bearing JJN3 or RPMI8226 xenografts, ARRY-520 administered alone was highly efficacious, with a 100% response rate (CR + PR) at doses of 12.5 to 20 mg/kg. Bortezomib administered alone at the maximum tolerated dose (MTD) of 1 mg/kg was only marginally efficacious (short term stable disease in JJN3 and H929), as was revlimid administered at 50 mg/kg (short term stable disease in RPMI8226). Because of the high initial response rate to ARRY-520 in these models, the best way to quantify the additional activity of combinations was to compare time to regrowth (time for tumor volume to return to the volume at initiation of treatment). As shown in the table, the combination of ARRY-520 with bortezomib or revlimid produced a significant increase in time to regrowth in several cases, compared to ARRY-520 alone. The additivity was schedule dependent, with greater efficacy observed when ARRY-520 was administered simulataneously with bortezomib, compared to ARRY-520 treatment before bortezomib. The activity of the ARRY-520 + bortezomib combination was specific for multiple myeloma models, as no additivity was observed in xenograft models of solid tumors (BxPC-3, LoVo, N87 and Hs294T). Conclusion: Combination of ARRY-520 with either bortezomib or revlimid has significantly greater activity than the single agents in xenograft models of multiple myeloma, suggesting these as rational combinations for evaluation in clinical trials. Disclosures: Woessner: Array BioPharma: Employment, Equity Ownership, Patents & Royalties. Tunquist:Array BioPharma: Employment, Patents & Royalties. Cox:Array BioPharma: Employment, Equity Ownership. Rana:Array BioPharma: Employment, Equity Ownership. Walker:Array BioPharma: Employment, Equity Ownership, Patents & Royalties. Lee:Array BioPharma: Employment, Equity Ownership, Patents & Royalties.

Beschreibung: Multiple Myeloma (MM) is a malignancy characterized by the clonal expansion of plasma cells within the bone marrow microenvironment. Within this compartment, the proliferative capacity of MM cells is enhanced by pro-inflammatory cytokines, and there is growing recognition that bone marrow stromal cells play an important role in the support of MM growth and chemo-resistance. We have identified a series of compounds, of which ARRY-614 is representative, that are potent inhibitors (EC50

Beschreibung: Kinesins are eukaryotic microtubule-associated motor proteins. One of these, kinesin spindle protein (KSP) is a mitotic-specific kinesin that plays a key role in spindle pole separation and production of the bipolar spindle, as well as centrosome separation and maturation. As KSP is expressed predominately in proliferating cells and is absent from postmitotic neurons, inhibition of KSP has the potential to provide the antitumor activity of a mitotic inhibitor while avoiding peripheral neuropathy. ARRY-429520 is a member of a series of KSP inhibitors discovered and optimized by structure-based design. It is a potent inhibitor of human KSP (IC50 6 nM), with an EC50 of 1.5 nM for cellular phosphorylation of histone H3 (a pharmacodynamic marker for accumulation of cells in mitosis), and in vitro antiproliferative IC50s ranging from subnanomolar to low single digit nanomolar across a varity of human and murine tumor cell lines. We report here the in vivo characterization of this compound. ARRY-429520 inhibits histone (H3) phosphorylation in tumor xenografts , showing that the compound has pharmacodynamic activity in vivo. As a result, it is a highly efficacious inhibitor of the growth of tumor xenografts. At its maximally tolerated dose in mice of 25 – 30 mg/kg i.p. on a Q4Dx3 schedule, ARRY-429520 caused tumor regressions, with some complete responses, in subcutaneous mouse xenograft models of leukemia: HL-60 (acute promyelocytic leukemia) and K-562 (chronic myelogenous leukemia). The compound was also highly efficacious against other tumor types, including HT-29 (colon), HCT-116 (colon), and A2780 (ovarian). ARRY-429520 also showed activity in intravenous leukemia models. The molecule possess desirable drug-like properties, including high aqueous solubility (〉 4 mg/ml at physiological pH), low CYP inhibition (〉 25uM for 3A4, 2C9, 1A2, 2D6, and 2C19), and pharmacokinetics favorable for an IV-infused targeted chemotherapeutic.