ALBERT

Description: Overexpression of the oncomiR, miR-155, is known to be predictive of poor outcome in chronic lymphocytic leukemia (CLL) patients. Using NanoString Technologies’ nCounter platform, we interrogated the miR-155 expression levels in 109 previously untreated CLL patients receiving chemoimmunotherapy on CALGB 9712 or CALGB 10101. The data, dichotomized around median expression, showed that high expressers of miR-155 had shorter progression-free survival (p=0.005) and a higher risk of death after 4 years on study (p=0.004). The expression of miR-155 was not significantly associated with the majority of baseline demographic, clinical and cytogenetic characteristics, including age, Rai stage and high-risk cytogenetics [del(17p)/del(11q)] (p〉0.15). Association of high miR-155 expression with IGHV un-mutated disease(p=0.03) and ZAP70 methylation

Description: In patients with chronic lymphocytic leukemia (CLL), lenalidomide can promote humoral immune responses but also induces a distinct disease-specific toxicity of tumor flare and cytokine release. These CLL-specific events result from increased expression of costimulatory molecules on B cells. Here we demonstrate that lenalidomide activation of CLL cells depends on the phosphatidylinositol 3-kinase p110δ (PI3K-δ) pathway. Inhibition of PI3K-δ signaling by the PI3K-δ-inhibiting drug, CAL-101, or by siRNA knockdown of p110δ, abrogates CLL cell activation, costimulatory molecule expression, and vascular endothelial growth factor and basic fibroblast growth factor gene expression that is induced by lenalidomide. In addition, CAL-101 attenuates lenalidomide-mediated increases in immunoglobulin M production by normal B cells. Collectively, these data demonstrate the importance of PI3K-δ signaling for lenalidomide immune modulation. These findings may guide development of strategies for the treatment of CLL that combine lenalidomide with CAL-101, with other inhibitors of the PI3K-δ pathway, or with other agents that target downstream kinases of this signaling pathway.

Description: Abstract 1821 Lenalidomide is an oral immune-modulating agent that has been shown to have clinical activity in patients with treatment-naive and previously treated chronic lymphocytic leukemia (CLL). In CLL, a disease-specific phenomenon of drug-induced tumor flare is often observed that results in lymph node enlargement, rash, and cytokine release. We and others have attributed both lenalidomide-induced tumor flare and cytokine release in part to CLL cell activation, with concomitant increase in surface co-stimulatory molecules including CD154. The potential consequences of such activation by lenalidomide in CLL are multiple. In symptomatic, previously untreated CLL, activation of tumor cells by lenalidomide likely contributes to reversal of hypogammaglobulinemia in a subset of patients. Additionally, activation of CLL cells increases their capacity for antigen presentation, potentially facilitating a clinically beneficial development of tumor-specific antibodies toward antigens such as ROR1. In patients with previously treated CLL, lenalidomide therapy does not reverse hypogammaglobulinemia. However, treatment has been documented to increase serum b-FGF and VEGF levels, which correlates with lack of response. Previous work demonstrates that CLL cells predominately utilize the PI3K p110δ isoform for activation following CD154 signaling. Given our prior findings of prominent lenalidomide induction of CD40-CD154 signaling in vitro and in vivo, we focused initially on molecular interrogation of isoforms responsible for this in CLL cells. Utilizing primary CLL cells, we demonstrated that inhibition of PI3K-δ signaling by CAL-101, a clinically relevant PI3K-δ isoform-specific inhibitor, abrogated lenalidomide-induced activation of CLL cells by directly reducing PI3K enzymatic activity and also reducing phosphorylation of the downstream PI3K target AKT. Parallel studies with siRNA targeted to the p110δ isoform of PI3K demonstrated antagonism of lenalidomide-induced AKT phosphorylation. Furthermore, we found that inhibition of PI3K-δ by CAL-101 at therapeutically relevant concentrations (1 μM) prevented up-regulation of CD40, CD154, and CD86 by lenalidomide and also antagonized production of IgM by normal B-cells co-cultured with CLL cells. Collectively, these data demonstrate the importance of PI3K-δ signaling in modulating the pharmacological effects of lenalidomide in CLL cell activation including up-regulation of CD40, CD154, CD86 and active CLL cell co-stimulation of normal B-cells. Our findings suggest that clinical evaluation of combination strategies of lenalidomide and CAL-101 in treatment-naive patients with CLL should be performed with careful pharmacodynamic monitoring of immune modulation and signaling to best preserve the clinical benefits of both drugs. This work is supported by the Leukemia and Lymphoma Society, D. Warren Brown Foundation, and The OSU Leukemia SPORE grant funded by the NCI. CAL-101 was provided by Calistoga Pharmaceuticals, Inc. Disclosures: Jones: Glaxo Smith-Kline: Consultancy; Abbott: Research Funding. Lannutti:Calistoga Pharmaceutical Inc.: Employment. Byrd:Calistoga Pharmaceutical Inc.: Equity Ownership. Johnson:Calistoga Pharmaceutical Inc.: Research Funding.

Description: Abstract 1385 Bruton tyrosine kinase (Btk), a member of the Tec family kinases, has a well-characterized role in B-cell antigen receptor (BCR) signaling and B-cell activation. Btk is activate by Src family kinases which lead to downstream activation of essential cell survival pathways such as NF-κB and MAP kinase. Although Btk is expressed in multiple hematopoietic cells, the primary defect in Btk knockout mice is B-cell specific, suggesting a more selective B-cell function consistent with its role in the BCR signaling pathway. Btk was identified in an unbiased screen as an essential signaling kinase for survival of certain lymphomas including diffuse large B-cell lymphoma, but no information is available about its role in CLL cell survival. Inhibition of Btk by PCI-32765, a selective irreversible Btk inhibitor, has demonstrated promising clinical activity in an ongoing phase 1 study in B-cell non-Hodgkin lymphoma. We therefore hypothesized that inhibition of Btk would induce cytotoxicity in B-CLL cells. BTK protein expression was observed in CLL cells, but not NK or T-cells as previously reported. Surprisingly, significant variability in BTK expression was noted among different CLL patient samples. Treatment of primary CLL cells with PCI-32765 at concentrations ranging from 0.1–10μM resulted in significant cell killing compared to untreated control. At a 10μM concentration, PCI-32765 resulted in a median 73% viable cells at 48 hours as detected by annexin V/propidium iodide flow cytometry (n=55). Although again, considerable variability was observed among patient samples (range 93.5–5.5%). We found no correlation between response to PCI-32765 and prognostic factors such as interphase cytogenetics or IgVH gene mutational status, suggesting a potential clinical benefit to even high-risk CLL patients. To determine the selectivity of PCI-32765, we evaluated cytotoxicity of lymphocytes derived from healthy volunteers. At 10μM, PCI-32765 resulted in a median 90% viable normal B-cells with little variability (n=5), showing substantially lower cytotoxicity in normal B-cells relative to CLL tumor cells. The cytotoxicity observed with PCI-32765 in CLL cells was accompanied by PARP cleavage and the induction of caspase 3 activity, and PCI-32765-mediated cell death was blocked by the pan-caspase inhibitor z-VAD-fmk (n=3). These results indicate that PCI-32765 is dependent on caspase activation for its ability to induce apoptosis in CLL cells. Further studies to determine the apoptotic mechanism showed that PCI-32765 treatment (10μM) partially reversed CD40L- or CpG-induced phosphorylation of ERK1/2 and also caused an increase in endoplasmic reticulum-derived intracellular calcium flux in CLL cells (n=8). Microenvironmental factors such as CD40L, TNF-α, IL-6 and BAFF can induce activation of CLL and/or decrease spontaneous apoptosis. Treatment of CLL cells with PCI-32765 prevented the protective anti-apoptotic effect promoted by these cytokines, suggesting that this agent can overcome the protection induced by microenvironmental stimuli. Similarly, co-culture of CLL cells on the human stromal cell line HS-5 significantly decreases the spontaneous apoptosis of CLL cells. PCI-32765 treatment was able to overcome this stromal protection, and induced apoptosis comparable to cells cultured in suspension (median cytotoxicity 86% vs. 84%, respectively; 10μM PCI-32765 at 48 hr, n=5). This suggests that the cytotoxic effect elicited by PCI-32765 will not be significantly diminished by the presence of an in vivo microenvironment. Taken together, our results suggest that PCI-32765 acts directly on CLL cells to induce apoptosis and helps to block microenvironment-derived protection. These investigations demonstrate the efficacy of targeting Btk in CLL and provide preclinical validation for ongoing Phase 1/2 clinical trials for the treatment of this disease. This work is supported by the Leukemia and Lymphoma Society, D. Warren Brown Foundation and The OSU Leukemia SPORE grant funded by the NCI. Disclosures: Buggy: Pharmacyclics, Inc.: Employment. Hamdy:Pharmacyclics, Inc.: Employment.

Description: B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell–specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted.

Description: Targeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101–mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell–activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders.