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Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and αIIbβ3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s–1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with αIIbβ3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of αIIbβ3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% ± 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti–phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s–1 but not at 200 s–1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block αIIbβ3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of αIIbβ3, is sufficient to induce SIPA but does not lead to stable thrombus formation.

Background PIM1, PIM2, and PIM3 are a closely related family of constitutively active serine/threonine kinases. PIM kinases were first identified as common integration sites in MMLV-induced murine T-cell lymphomas, with these viral insertions resulting in transcriptional activation and expression of the kinase. Similar unbiased insertional mutagenesis screens were used to demonstrate that the three kinases could substitute for each other in this oncogenic process, indicating that inhibition of all three PIM kinases may be necessary to achieve a therapeutic benefit. In human cancers, increased expression and activity of PIM kinases has been detected in many hematopoietic malignancies and solid tumors (prostate, lung, liver) and it is thought that this contributes to cancer cell survival and proliferation in these malignancies. Methods & Results We have developed a potent and specific pan-PIM inhibitor, LGH447, and shown that it is active in PIM2-dependent Multiple Myeloma (MM) cell lines by inhibiting proliferation, mTOR-C1 signaling and phosphorylation of BAD. In addition, LGH447 inhibited proliferation of several AML cell lines. Furthermore, we demonstrated that LGH447 inhibits tumor growth in several mouse subcutaneous xenograft models of MM and AML, where modulation of the pharmacodynamic marker phospho-S6 Ribosomal Protein was used to predict efficacy. We have also determined that LGH447 significantly reduced the bone tumor burden in a disseminated orthotopic human xenograft model of MM. Finally, we have demonstrated increased activity of LGH447 in combination with the PI3K inhibitor BYL719 in a MM model and with Cytarabine in an AML model. Summary Our Results have supported the clinical development of LGH447, a Pan-PIM inhibitor, in relapsed, refractory MM, and further suggest that it may be effective in the treatment of AML and other hematological malignancies, either as a single agent or in combination with other select therapeutic agents. Disclosures: Garcia: Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Langowski:Novartis Institutes for Biomedical Research: Employment. Holash:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Burger:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Zang:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Zavorotinskaya:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Fanton:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Saci:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Growney:Novartis Institutes for Biomedical Research: Employment, Equity Ownership. Vanasse:Novartis Pharmaceuticals Corporation: Employment.

Background The JAK-STAT pathway is an important signaling pathway downstream of multiple cytokine and growth factor receptors. It has been implicated in the pathogenesis of multiple human diseases. The genetic aberration of JAK2V617F and the associated activation of STAT in myeloproliferative neoplasia (MPN) is one example of the involvement of this pathway in human cancer. Activated JAKs phosphorylate STAT proteins, which then dimerize and translocate to the nucleus. Inside the nucleus, activated STATs modulate the expression of target genes, including PIM1. PIM kinases are involved in the regulation of cell cycle and proliferation. Recent studies have suggested that PIM1 is one of the key signature genes for STAT5 activation. Based on the biological rationale, we set out to test the combination of the JAK inhibitor, ruxolitinib, and the PIM inhibitor, LGH447, in MPN preclinical models. Methods and Results The combination of ruxolitinib and LGH447 was tested in vitro against MPN cell lines harboring JAK2V617F mutations. In vitro synergy was observed with the combination of ruxolitinib and LGH447 in UKE-1 and SET2 models. Pharmacodynamic analysis indicates potent modulation of PIM1/2 and pSTAT3/5 by LGH447 and ruxolitinib, respectively. Both agents inhibited pERK and pS6 in UKE-1. The combination achieved greater suppression on these two signaling pathways. In addition, the combination further inhibited pBAD and MCL-1 expression, with the corresponding increase of cleaved PARP. This combination was further tested in a mouse MPN model with BA/F3 cells harboring EPOR-JAK2V617F. Ruxolitinib monotherapy down-modulated overall disease burden and reduced spleen weight. It also inhibited pSTAT5 and pMEK in this model. LGH447 monotherapy had a more modest effect on MPN disease burden. The combination of ruxolitinib and LGH447 resulted in much greater reduction of overall disease burden and spleen weight than either agent alone (Figure). The combination also normalized hemoglobin levels and red blood cell counts in this model. Summary This finding suggests potential benefit with the combination of ruxolitinib and LGH447 in MPN. By targeting two critical signaling molecules in the JAK/STAT axis, this combination may achieve greater target modulation across key oncogenic pathways, such as MAPK and TOR. This may enable us to achieve greater benefit against MPN in the clinic. Disclosures: Saci: Novartis: Employment, Equity Ownership. Pinzon-Ortiz:Novartis Institutes for BioMedical Research, Inc.: Employment. Wang:Novartis: Employment. Rong:Novartis: Employment. Growney:Novartis: Employment, Equity Ownership. Squier:Novartis Pharmaceuticals Corp.: Employment. Radimerski:Novartis Pharma AG: Employment, Equity Ownership. Vanasse:Novartis Institutes of BioMedical Research: Employment. Caponigro:Novartis: Employment, Equity Ownership. Garcia:Novartis: Employment, Equity Ownership. Cao:Novartis: Employment, Equity Ownership.