Leslie A. Andritsos

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.

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

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.

Higher Doses of Lenalidomide Are Associated With Unacceptable Toxicity Including Life-Threatening Tumor Flare in Patients With Chronic Lymphocytic Leukemia

PURPOSE Lenalidomide is a novel therapeutic agent with uncertain mechanism of action that is clinically active in myelodysplastic syndrome (MDS) and multiple myeloma (MM). Application of high (MM) and low (MDS) doses of lenalidomide has been reported to have clinical activity in CLL. Herein, we highlight life-threatening tumor flare when higher doses of lenalidomide are administered to patients with CLL and provide a potential mechanism for its occurrence. PATIENTS AND METHODS Four patients with relapsed CLL were treated with lenalidomide (25 mg/d for 21 days of a 28-day cycle). Serious adverse events including tumor flare and tumor lysis are summarized. In vitro studies examining drug-induced apoptosis and activation of CLL cells were also performed. RESULTS Four consecutive patients were treated with lenalidomide; all had serious adverse events. Tumor flare was observed in three patients and was characterized by dramatic and painful lymph node enlargement resulting in hospitalization of two patients, with one fatal outcome. Another patient developed sepsis and renal failure. In vitro studies demonstrated lenalidomide-induced B-cell activation (upregulation of CD40 and CD86) corresponding to degree of tumor flare, possibly explaining the tumor flare observation. CONCLUSION Lenalidomide administered at 25 mg/d in relapsed CLL is associated with unacceptable toxicity; the rapid onset and adverse clinical effects of tumor flare represent a significant limitation of lenalidomide use in CLL at this dose. Drug-associated B-cell activation may contribute to this adverse event. Future studies with lenalidomide in CLL should focus on understanding this toxicity, investigating patients at risk, and investigating alternative safer dosing schedules.

Lenalidomide treatment promotes CD154 expression on CLL cells and enhances production of antibodies by normal B cells through a PI3-kinase–dependent pathway

Chronic lymphocytic leukemia (CLL) involves a profound humoral immune defect and tumor-specific humoral tolerance that directly contribute to disease morbidity and mortality. CD154 gene therapy can reverse this immune defect, but attempts to do this pharmacologically have been unsuccessful. The immune-modulatory agent lenalidomide shows clinical activity in CLL, but its mechanism is poorly understood. Here, we demonstrate that lenalidomide induces expression of functional CD154 antigen on CLL cells both in vitro and in vivo. This occurs via enhanced CD154 transcription mediated by a Nuclear Factor of Activated T cells c1 (NFATc1)/Nuclear Factor-kappaB (NF-kappaB) complex and also through phosphoinositide-3 (PI3)-kinase pathway-dependent stabilization of CD154 mRNA. Importantly, CD154-positive CLL cells up-regulate BID, DR5, and p73, become sensitized to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis, and promote costimulatory activation of normal B cells to produce antibodies. In CLL patients receiving lenalidomide, similar evidence of CD154 activation is observed including BID, DR5, and p73 induction and also development of anti-ROR1 tumor-directed antibodies. Our data demonstrate that lenalidomide promotes CD154 expression on CLL cells with subsequent activation phenotype, and may therefore reverse the humoral immune defect observed in this disease. This study is registered at http://clinicaltrials.gov as NCT00466895.

Lenalidomide down-regulates the CD20 antigen and antagonizes direct and antibody-dependent cellular cytotoxicity of rituximab on primary chronic lymphocytic leukemia cells

Lenalidomide, an immunomodulatory agent that enhances antibody-dependent cellular cytotoxicity (ADCC), is currently being investigated as a therapy for chronic lymphocytic leukemia (CLL). The anti-CD20 antibody rituximab is active in CLL and represents a rational agent to combine with lenalidomide. We therefore examined whether lenalidomide combined with rituximab enhances direct apoptosis and ADCC in CLL cells. In contrast to previous reports using CD20-positive lymphoma cell lines, lenalidomide down-regulated CD20 surface antigen expression in CLL patient cells via enhanced internalization, without influencing transcription. The CD20 surface antigen internalization enhanced delivery of an oligonucleotide incorporated into anti-CD20 immunoliposomes. In addition, CD20 surface antigen down-modulation by lenalidomide in CLL was accompanied by diminished rituximab-mediated apoptosis and ADCC. These observations suggest a need for alternative sequencing strategies to avoid antagonism between lenalidomide and rituximab therapy in CLL. In addition, they suggest that lenalidomide therapy might be useful to enhance targeted delivery of RNAi-based therapies using CD20 immunoliposomes in B-cell malignancies.

Safety and activity of BTK inhibitor ibrutinib combined with ofatumumab in chronic lymphocytic leukemia: a phase 1b/2 study.

Ibrutinib represents a therapeutic advance in chronic lymphocytic leukemia (CLL) but as monotherapy produces few complete remissions in previously treated patients. Anti-CD20 antibodies have improved response and progression-free survival (PFS) when combined with chemotherapy. We evaluated the safety and activity of adding ofatumumab to ibrutinib in 3 different administration sequences. Patients with CLL/small lymphocytic lymphoma (SLL), prolymphocytic leukemia, or Richters transformation who failed ≥2 prior therapies were enrolled. Patients received ibrutinib 420 mg daily and 12 doses of ofatumumab 300/2000 mg in 3 schedules: ibrutinib lead-in (group 1; n = 27), concurrent start (group 2; n = 20), or ofatumumab lead-in (group 3; n = 24). Seventy-one patients were treated; most had high-risk disease including del(17)(p13.1) (44%) or del(11)(q22.3) (31%). The most frequent adverse events (any grade) were diarrhea (70%), infusion-related reaction (45%), and peripheral sensory neuropathy (44%). Overall response rates in CLL/SLL patients (n = 66) were 100%, 79%, and 71% in groups 1, 2, and 3, respectively. Estimated 12-month PFSs for all patients were 89%, 85%, and 75%, respectively. Four patients in group 3 progressed prior to receiving ibrutinib. This study demonstrates the tolerability and clinical activity of this combination with quicker time to best response than single-agent ibrutinib and with durable responses. This trial was registered at www.clinicaltrials.gov as #NCT01217749.

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.

Improved nonrelapse mortality and infection rate with lower dose of antithymocyte globulin in patients undergoing reduced-intensity conditioning allogeneic transplantation for hematologic malignancies.

We sought to reduce the risk of infectious complications and nonrelapse mortality (NRM) associated with the use of antithymocyte globulin (ATG) without compromising control of acute graft-versus-host disease (aGVHD) in patients undergoing reduced-intensity conditioning (RIC) transplantation. As part of an ongoing quality improvement effort, we lowered the dose of rabbit ATG from 7.5 mg/kg of ATG (R-ATG) (n = 39) to 6.0 mg/kg of ATG (r-ATG) (n = 33) in association with fludarabine (Flu) and busulfan (BU) RIC transplantation and then monitored patients for adverse events, relapse, and survival. Of the 72 mostly high risk (82%) patients studied, 89% received unrelated donor allografts, 25% of which were HLA-mismatched. No differences in posttransplantation full donor-cell chimerism rates were observed between the 2 ATG-dose groups (P > .05). When R-ATG versus r-ATG patients were compared, we observed no significant difference in the cumulative incidence of grade II-IV aGVHD (32% versus 27%; P = .73) or grade III-IV aGVHD (23% versus 11%; P = .28). However, the r-ATG group had significantly less cytomegalovirus (CMV) reactivation (64% versus 30%; P = .005) and bacterial infections (56% versus 18%; P = .001), a better 1-year cumulative incidence of NRM (18% versus 3%; P = .03), and a trend for better 1-year overall survival (OS) (64% versus 84%; P = .07) compared to R-ATG patients. A seemingly modest reduction in the dose of rabbit ATG did not compromise control of aGVHD or achievement of donor chimerism, but led to a significant decrease in the risk of serious infections and NRM in high-risk RIC allograft recipients.

Hypermorphic mutation of phospholipase C, γ2 acquired in ibrutinib-resistant CLL confers BTK independency upon B-cell receptor activation

Ibrutinib has significantly improved the outcome of patients with relapsed chronic lymphocytic leukemia (CLL). Recent reports attribute ibrutinib resistance to acquired mutations in Bruton agammaglobulinemia tyrosine kinase (BTK), the target of ibrutinib, as well as the immediate downstream effector phospholipase C, γ2 (PLCG2). Although the C481S mutation found in BTK has been shown to disable ibrutinibs capacity to irreversibly bind this primary target, the detailed mechanisms of mutations in PLCG2 have yet to be established. Herein, we characterize the enhanced signaling competence, BTK independence, and surface immunoglobulin dependence of the PLCG2 mutation at R665W, which has been documented in ibrutinib-resistant CLL. Our data demonstrate that this missense alteration elicits BTK-independent activation after B-cell receptor engagement, implying the formation of a novel BTK-bypass pathway. Consistent with previous results, PLCG2(R665W) confers hypermorphic induction of downstream signaling events. Our studies reveal that proximal kinases SYK and LYN are critical for the activation of mutant PLCG2 and that therapeutics targeting SYK and LYN can combat molecular resistance in cell line models and primary CLL cells from ibrutinib-resistant patients. Altogether, our results engender a molecular understanding of the identified aberration at PLCG2 and explore its functional dependency on BTK, SYK, and LYN, suggesting alternative strategies to combat acquired ibrutinib resistance.