Amy J. Wagner

Phosphatidylinositol 3-kinase-δ inhibitor CAL-101 shows promising preclinical activity in chronic lymphocytic leukemia by antagonizing intrinsic and extrinsic cellular survival signals.

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.

Phase II Study of Flavopiridol in Relapsed Chronic Lymphocytic Leukemia Demonstrating High Response Rates in Genetically High-Risk Disease

PURPOSE Patients with chronic lymphocytic leukemia (CLL) with high-risk genomic features achieve poor outcomes with traditional therapies. A phase I study of a pharmacokinetically derived schedule of flavopiridol suggested promising activity in CLL, irrespective of high-risk features. Given the relevance of these findings to treating genetically high-risk CLL, a prospective confirmatory study was initiated. PATIENTS AND METHODS Patients with relapsed CLL were treated with single-agent flavopiridol, with subsequent addition of dexamethasone to suppress cytokine release syndrome (CRS). High-risk genomic features were prospectively assessed for response to therapy. RESULTS Sixty-four patients were enrolled. Median age was 60 years, median number of prior therapies was four, and all patients had received prior purine analog therapy. If patients tolerated treatment during week 1, dose escalation occurred during week 2. Dose escalation did not occur in four patients, as a result of severe tumor lysis syndrome; three of these patients required hemodialysis. Thirty-four patients (53%) achieved response, including 30 partial responses (PRs; 47%), three nodular PRs (5%), and one complete response (1.6%). A majority of high-risk patients responded; 12 (57%) of 21 patients with del(17p13.1) and 14 (50%) of 28 patients with del(11q22.3) responded irrespective of lymph node size. Median progression-free survival among responders was 10 to 12 months across all cytogenetic risk groups. Reducing the number of weekly treatments per cycle from four to three and adding prophylactic dexamethasone, which abrogated interleukin-6 release and CRS (P < or = .01), resulted in improved tolerability and treatment delivery. CONCLUSION Flavopiridol achieves significant clinical activity in patients with relapsed CLL, including those with high-risk genomic features and bulky lymphadenopathy. Subsequent clinical trials should use the amended treatment schedule developed herein and prophylactic corticosteroids.

17-DMAG targets the nuclear factor-κB family of proteins to induce apoptosis in chronic lymphocytic leukemia: clinical implications of HSP90 inhibition

The HSP90 client chaperone interaction stabilizes several important enzymes and antiapoptotic proteins, and pharmacologic inhibition of HSP90 results in rapid client protein degradation. Therefore, HSP90 inhibition is an attractive therapeutic approach when this protein is active, a phenotype commonly observed in transformed but not normal cells. However, preclinical studies with HSP90 inhibitors such as 17-AAG demonstrated depletion of only a subset of client proteins and very modest tumor cytotoxicity in chronic lymphocytic leukemia (CLL) cells. Herein, we describe another HSP90 inhibitor, 17-DMAG, which is cytotoxic to CLL but not normal lymphocytes. Treatment with 17-DMAG leads to depletion of the HSP90 client protein IKK, resulting in diminished NF-kappaB p50/p65 DNA binding, decreased NF-kappaB target gene transcription, and caspase-dependent apoptosis. Furthermore, treatment with 17-DMAG significantly decreased the white blood cell count and prolonged the survival in a TCL1-SCID transplant mouse model. The ability of 17-DMAG to function as an NF-kappaB inhibitor is of great interest clinically, as few currently available CLL drugs target this transcription factor. Therefore, the effect of 17-DMAG on NF-kappaB signaling pathways represents a novel therapy warranting further clinical pursuit in this and other B-cell lymphoproliferative disorders.

ER stress and autophagy: new discoveries in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol

Cyclin dependent kinase (CDK) inhibitors, such as flavopiridol, demonstrate significant single-agent activity in chronic lymphocytic leukemia (CLL), but the mechanism of action in these nonproliferating cells is unclear. Here we demonstrate that CLL cells undergo autophagy after treatment with therapeutic agents, including fludarabine, CAL-101, and flavopiridol as well as the endoplasmic reticulum (ER) stress-inducing agent thapsigargin. The addition of chloroquine or siRNA against autophagy components enhanced the cytotoxic effects of flavopiridol and thapsigargin, but not the other agents. Similar to thapsigargin, flavopiridol robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases. Both autophagy and ER stress were documented in tumor cells from CLL patients receiving flavopiridol. Thus, CLL cells undergo autophagy after multiple stimuli, including therapeutic agents, but only with ER stress mediators and CDK inhibitors is autophagy a mechanism of resistance to cell death. These findings collectively demonstrate, for the first time, a novel mechanism of action (ER stress) and drug resistance (autophagy) for CDK inhibitors, such as flavopiridol in CLL, and provide avenues for new therapeutic combination approaches in this disease.

The novel cyclin-dependent kinase inhibitor dinaciclib (SCH727965) promotes apoptosis and abrogates microenvironmental cytokine protection in chronic lymphocytic leukemia cells

The cyclin dependent kinase (CDK) inhibitor flavopiridol has demonstrated promising clinical results in relapsed CLL patients leading to efforts to develop improved CDK inhibitors. Dinaciclib (SCH727965) is a pan-CDK inhibitor, derived from a detailed screen in ovarian xenograft mouse models for therapeutic index, whose toxicity in solid tumor phase I studies appears favorable. Dinaciclib in CLL cells demonstrates concentration dependent apoptosis that is superior to flavopiridol following a clinically relevant 2-hour exposure. Dinaciclib potently down-regulates expression of Mcl-1 in CLL cells and antagonizes protection mediated by multiple soluble proteins important in the microenvironment of CLL including TNF-α IL-4, BAFF, and CD40-ligand. In contrast, contact with stromal cells or fibronectin abrogates the cytotoxicity of dinaciclib that is antagonized by a pan inhibitor and p110 alpha isoform specific inhibitor of the phosphatidylinositol 3-kinase pathway suggesting potential for combination strategies. These data justify clinical development of dinaciclib in CLL.

Rituximab and 17-allylamino-17-demethoxygeldanamycin induce synergistic apoptosis in B-cell chronic lymphocytic leukaemia

Treatment options for chronic lymphocytic leukaemia (CLL) are limited and eventually fail because of the development of toxicities or drug resistance. Thus, identification of new therapeutic strategies and targets is a high priority. The semisynthetic geldanamycin derivative 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) inhibits heat shock protein 90 (Hsp90) binding to client proteins, thereby leading to their degradation. We demonstrate that at biologically active and clinically attainable levels (1 μmol/l), 17‐AAG treatment of CLL B cells in vitro causes modest apoptosis as well as decreased AKT protein levels. Given the potential activation of AKT following antibody therapy in CLL, we evaluated the combination of 17‐AAG and rituximab. These agents produced synergistic cytotoxicity of CLL cells in vitro. However, rituximab‐mediated antibody‐dependent cellular cytotoxicity was modestly reduced with 17‐AAG, and complement‐dependent cytotoxicity was not altered. We conclude that the combination of Hsp90 inhibitors with therapeutic antibodies, such as rituximab may represent a novel strategy to enhance therapeutic response in CLL. Furthermore, our data indicates that AKT and Hsp70 protein levels are relevant pharmacodynamic endpoints to monitor the in vivo effect of 17‐AAG therapy.

A phase I trial of the intravenous Hsp90 inhibitor alvespimycin (17-DMAG) in patients with relapsed chronic lymphocytic leukemia/small lymphocytic lymphoma.

Despite recent advances in treatment for patients with chronic lymphocytic leukemia (CLL), the disease remains incurable with approved therapies outside of allogeneic stem cell transplantation. Unf...