Key Points• AEB071 demonstrates preclinical in vitro and in vivo activity against CLL independent of survival signaling and stromal cell protection.• AEB071 can either inhibit or activate the WNT pathway emphasizing the importance of pharmacodynamic monitoring in its development.Targeting B-cell receptor (BCR) signaling in chronic lymphocytic leukemia (CLL) has been successful with durable remissions observed with several targeted therapeutics. Protein kinase C-b (PKC-b) is immediately downstream of BCR and has been shown to be essential to CLL cell survival and proliferation in vivo. We therefore evaluated sotrastaurin (AEB071), an orally administered potent PKC inhibitor, on CLL cell survival both in vitro and in vivo. AEB071 shows selective cytotoxicity against B-CLL cells in a dose-dependent manner. Additionally, AEB071 attenuates BCR-mediated survival pathways, inhibits CpG-induced survival and proliferation of CLL cells in vitro, and effectively blocks microenvironment-mediated survival signaling pathways in primary CLL cells. Furthermore, AEB071 alters b-catenin expression, resulting in decreased downstream transcriptional genes as c-Myc, Cyclin D1, and CD44. Lastly, our preliminary in vivo studies indicate beneficial antitumor properties of AEB071 in CLL. Taken together, our results indicate that targeting PKC-b has the potential to disrupt signaling from the microenvironment contributing to CLL cell survival and potentially drug resistance. Future efforts targeting PKC with the PKC inhibitor AEB071 as monotherapy in clinical trials of relapsed and refractory CLL patients are warranted. (Blood. 2014;124(9):1481-1491) IntroductionChronic lymphocytic leukemia (CLL) is one of the most common types of adult leukemia and is currently incurable. Decades of research into the biology of CLL and other B-cell malignancies has brought forth B-cell receptor (BCR) signaling as a common required driving force in the survival and proliferation of these tumor cells. Several survival pathways involved in BCR signaling, including the phosphatidylinositol 3-kinase (PI3K), nuclear factor-kB (NF-kB), and mitogen-activated protein kinase (MAPK)/extracellular signalregulated kinase (ERK), are constitutively active in the lymph node and bone marrow compartment of CLL where disease expansion occurs. [1][2][3] Efforts to target BCR signaling with therapeutic agents which reversibly inhibit PI3K-d 4,5 or irreversibly inhibit Bruton tyrosine kinase (BTK) 6-8 have shown significant clinical activity in CLL, including those with high-risk genomic disease, and are currently in phase 3 studies. Protein kinase C-b (PKC-b) is an immediate downstream target of BTK that has recently been shown to be overexpressed in CLL 9 and is essential to the in vivo development of CLL in Em-TCL1 mice. 10 In B cells, PKC-b is thought to be the predominant PKC isoform mediating BCRdependent NF-kB activation.11-13 Downstream of PKC-b, IkB kinase and caspase recruitment domain-containing protein 11 (CARD11 [also known as CARMA1]) are phosphorylated, le...

Acute Myeloid Leukemia (AML) is the most common adult acute leukemia and is characterized by numerous driver mutations and/or cytogenetic rearrangements that promote disruption of stem cell/early myeloid progenitor differentiation, apoptosis, and proliferation. Identification of both personalized targets specific to a mutation or genomic abnormality and also global ubiquitous tumor-related targets relevant to AML represents a high priority to improve therapy. p21 protein (Cdc42/Rac)-activated kinase 4 (PAK4) is involved in disease progression for several solid tumors but its expression and contribution to disease pathogenesis in AML has not been examined. Since multiple cellular pathways important in AML (e.g., RAS and Wnt/β-catenin) are regulated by PAKs, we hypothesized PAK4 (and other family members) could represent an attractive pharmacologic target. We first evaluated the expression of PAK4 in AML cell lines and patient blasts (55 patients) using RNA sequencing and Western blot confirmation. This demonstrated PAK4 to be abundantly expressed at the mRNA and protein level in virtually all the analyzed samples. We then tested the in vitro effects of the PAK4 allosteric modulators (PAMs) KPT-9274 (clinical candidate) and KPT-9331 (tool compound) on AML cell lines. These included MV4-11, HL-60, THP-1 and Kasumi-1. Cell lines were treated for 24, 48 and 72 with PAMs KPT-9274 and KPT-9331 at dosages ranging from 1nM to 10uM. Proliferation was measured by MTS assay. All cell lines showed a dose-and time- dependent decrease in cell proliferation with IC50 ranging from 0.14 to 0.28 µM for both compounds. Cell lines with low protein and mRNA expression of PAK4, such as HL-60, were sensitive to PAM treatment suggesting possible alternative targets of these agents. To determine the effect of PAMs on apoptosis, MV4-11 and THP-1 cell lines were treated with KPT-9331 at IC50 concentration (~0.25 µM) and cell death was measured via Annexin-V/PI flow cytometric analysis after 24, 48h and 72h treatment. KPT-9331 induced a time dependent increase in apoptosis in both cell lines. In MV4-11 cells, KPT-9331 caused cell cycle arrest and inhibition of proliferation after 24hrs. We also tested the effect of PAMs in primary AML cells. Patient samples cocultured with a human stromal cell lines were treated with PAMs for 96 hours. IC50 values ranged from 0.14 - 0.19 µM. A dose dependent decrease in proliferation following PAM treatment was observed in all the five analyzed samples irrespective of genetic subtype. PAMs treatment for 48hrs using a whole blood viability assay from normal donors showed no significant cytotoxic effect on T and NK cells, but modest toxicity to normal B cells. Normal hematopoietic colony forming cell assays are being initiated and will be presented. We next utilized a human AML leukemia xenograft model with MV4-11 cells to assess the in vivo activity of KPT-9274. Mice were dosed once daily via oral gavage with KPT-9274 (150 mg/kg) or vehicle control. KPT-9274 dramatically inhibited tumor growth, prevented invasion of MV4-11 cells, and improved overall survival with all mice (n=7) being alive (median not reached) at day 49 of experiment as compared to 1 out of 7 vehicle-treated mice (median survival 36 days) being alive at this time. In summary, KPT-9274 demonstrates promising activity in pre-clinical AML models and warrants further investigation in this disease. Ongoing efforts include validating the specificity of the reported target in AML (versus alternative targets), in vivo exploration in primary human AML xenograft models, and understanding the effects of this compound on normal hematopoiesis and function. Disclosures Baloglu: Karyopharm Therapeutics Inc.: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Blum:Gilead Sciences: Research Funding. Byrd:Acerta Pharma BV: Research Funding.

Richter's syndrome (RS) represents the transformation of B-cell chronic lymphocytic leukemia (CLL) to a large cell or immunoblastic lymphoma occurring in up to 15% of patients and is associated with poor prognosis and limited treatment options. While RS was first described in 1928, the molecular, genetic and/or epigenetic events that drive CLL B-cell transformation to lymphoma remain poorly characterized. A more comprehensive understanding of the pathogenesis underlying CLL disease progression to lymphoma is needed to (i) reveal potential biomarker(s) to identify CLL patients at risk of transformation and (ii) facilitate the discovery of novel targeted therapeutic approaches for this incurable disease. Epigenetic regulation mediated by lysine- and arginine modifying enzymes play an essential role in tumorigenesis and enhanced nuclear expression of the type II protein arginine methyltransferase, PRMT5, has been associated with increased cell proliferation and survival. Studies on PRMT5 demonstrate its function as an oncogene and its involvement in epigenetic silencing of tumor suppressor and growth regulatory genes in tumor cells; however, its implication in CLL progression is not known. Immunoblot analysis of PRMT5 expression in CLL cells derived from 50 CLL patients with low- (13q) or high-risk (del11q and/or 17p) cytogenetic features revealed no differences in PRMT5 expression among the different cytogenetic groups. However, evaluation of the cellular localization of PRMT5 in 28 patients revealed significantly elevated nuclear PRMT5 levels in CLL cells derived from the high-risk patients. We next evaluated the expression of PRMT5 in CLL cells from patients that developed RS and demonstrate that PRMT5 is overexpressed months to years prior to transformation. This suggests a possible role of PRMT5 overexpression as both a driver event in CLL transformation to DLBCL as well as a potential therapeutic target for intervention. Inhibition of PRMT5 using two small molecule, selective inhibitors (HLCL-61 and HLCL-65) was tested on CLL cells and a significant correlation was observed between HLCL-65 induced cytotoxicity and PRMT5 expression in primary CLL tumor cells. However when patients were separated in low- and high-risk disease groups a significant correlation was observed only in the high-risk group. The effect of inhibition of PRMT5 was also tested in presence or absence of CpG oligonucleotides, a well characterized inducer of CLL cell proliferation. PRMT5 inhibition resulted in significant cytotoxicity and attenuated the survival effect induced by CpG stimulation in CLL cells derived from patients with either low- or high-risk disease. Notably, PRMT5 inhibition markedly interfered with critical pathways important for CLL progression and survival including PI3K, MAPK/ERK as well as cMyc and cJun in CpG-stimulated cells. Additionally, nuclear PRMT5 levels were found to be markedly higher in CpG-stimulated B-cells suggesting a possible role of PRMT5 in the CpG-induced survival pathways. The expression pattern of PRMT5 was also studied in the Eμ-TCL1 transgenic mouse model of CLL. Immunoblot analysis of splenocytes derived from mice at different disease stage revealed a higher level of nuclear PRMT5 in mice with advanced disease. Notably, PRMT5 inhibition in B-cells derived from mice with advanced leukemia induced significant cytotoxicity and reexpression of ST7 a known PRMT5 target gene as well as derepression of miRs implicated in malignant B-cells such as mir-16, miR-106b and miR-223. Together, these findings indicate that PRMT5 contributes to CLL disease progression, and may prove as a candidate prognostic marker identifying CLL patients at risk for transformation; wherein therapeutic strategies aimed at inhibiting PRMT5-driven oncogenic pathways are highly warranted. Disclosures No relevant conflicts of interest to declare.

Chronic Lymphocytic Leukemia (CLL) is a B-cell malignancy with aberrant activation of the B-cell receptor (BCR) pathway. Despite durable remissions with targeted therapies (e.g., ibrutinib) in CLL, it remains an incurable disease. Epigenetic modifications, including DNA methylation and dysregulation of chromatin regulators have been shown to contribute to the neoplastic phenotype and the differential biologic behavior of tumor cells, including leukemia. An additional layer of epigenetic complexity in cancer cells is the acquisition of super-enhancer regions enriched at genes with known oncogenic function including MYC and BCL2. Super-enhancers in multiple myeloma cells and other tumors have been found strongly enriched for binding of BRD4, a member of the human bromodomain and extraterminal (BET) domain family of proteins which includes BRD2, BRD3, BRD4, and the testis-specific member BRDT. BRD4 binds to acetylated lysines on histones and regulates the expression of important oncogenes (e.g., MYC and BCL2). We investigated the therapeutic benefit of BET inhibition in cell culture and in vivo disease models of leukemia/lymphoma using PLX51107, a novel BRD4 inhibitor with unique binding mode. Results: We report that BRD4 is significantly overexpressed in CLL patient-derived B-cells compared to B-cells from healthy donors on both transcript and protein level (p < .001). RNA-seq analysis of 55 CLL patients revealed expression of various BRD4 isoforms with marked abundance of BRD4-long and BRD4-short. Next we sought to investigate the anti-tumor activity of PLX51107 in multiple malignant B-cell lines and patient-derived CLL cells. PLX51107 inhibited cell growth in MEC1, OCI-Ly2 and OCI-Ly6 (p < .001) dose-dependently with IC50 of 1.0 ± 0.09, 1.2 ± 0.05, 1.8 ± 0.05 μM, respectively. Notably, PLX51107 antagonized CpG-induced increase in cell proliferation of primary CLL cells (p < .01) which was consistent with the downmodulation of MYC and MCL1 along with the accumulation of the cyclin-dependent kinase inhibitor p21 and IκBα (p < .005). Furthermore, the efficacy of PLX51107 to disrupt survival signaling from the microenvironment was investigated under co-culture conditions with two different bone marrow stroma cell lines, wherein PLX51107 treatment significantly induced cytotoxicity in B-CLL cells (p < .01) without affecting stromal cell viability. By employing microarray analysis we identified possible novel targets of BRD4 in CLL. Validation of those targets is currently ongoing. Particularly, Bruton's tyrosine kinase (BTK) and phospholipase C gamma 2 (PLCG2) were markedly decreased with PLX51107 treatment (p < .005), thereby signifying potential therapeutic effect(s) for dual targeting of BRD4 and BCR-associated kinases to achieve deeper and durable responses in relapsed/refractory B-cell malignancies. Lastly, anti-tumor effects of BRD4 inhibition were evaluated in vivo using Eμ-TCL1 and cMYC/TCL1 adoptive transfer models of leukemia and lymphoma, respectively. In the Eμ-TCL1 engraftment model of aggressive CLL, PLX51107 treatment resulted in prolonged survival (p < .001) accompanied with decreased disease burden, lymphocyte infiltration and proliferation when compared to vehicle-treated mice. Next, the cMYC/TCL1 adoptive transfer mouse model was used to evaluate BRD4 inhibition in a highly penetrant, malignant leukemia/lymphoma phenotype analogous to high grade lymphoma wherein PLX51107 prolonged survival (p < .0001), decreased peripheral lymphocyte counts and neoplastic cell infiltration and proliferation in both spleen and lymph nodes. Conclusion: Collectively our findings reveal BRD4 as a valid and novel target for epigenetic therapy directed against core transcriptional programs in malignant/proliferating B-cells and provide support for use of PLX51107 as an effective treatment in clinical trials for relapsed/refractory CLL patients and related aggressive forms of B-cell malignancies, with the ultimate goal of improving the outcome of these patients. Disclosures Byrd: Acerta Pharma BV: Research Funding.