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Bruton’s tyrosine kinase (BTK) is a critical mediator of survival in B-cell neoplasms. Although BTK inhibitors have transformed therapy in chronic lymphocytic leukemia (CLL), patients with high-risk genetics are at risk for relapse and have a poor prognosis. Identification of novel therapeutic strategies for this group of patients is an urgent unmet clinical need, and therapies that target BTK via alternative mechanisms may fill this niche. Herein, we identify a set of microRNAs (miRs) that target BTK in primary CLL cells and show that the histone deacetylase (HDAC) repressor complex is recruited to these miR promoters to silence their expression. Targeting the HDACs by using either RNA interference against HDAC1 in CLL or a small molecule inhibitor (HDACi) in CLL and mantle cell lymphoma restored the expression of the BTK-targeting miRs with loss of BTK protein and downstream signaling and consequent cell death. We have also made the novel and clinically relevant discovery that inhibition of HDAC induces the BTK-targeting miRs in ibrutinib-sensitive and resistant CLL to effectively reduce both wild-type and C481S-mutant BTK. This finding identifies a novel strategy that may be promising as a therapeutic modality to eliminate the C481S-mutant BTK clone that drives resistance to ibrutinib and provides the rationale for a combination strategy that includes ibrutinib to dually target BTK to suppress its prosurvival signaling.

Despite the therapeutic efficacy of ibrutinib in chronic lymphocytic leukemia (CLL), complete responses are infrequent, and acquired resistance to Bruton agammaglobulinemia tyrosine kinase (BTK) inhibition is being observed in an increasing number of patients. Combination regimens that increase frequency of complete remissions, accelerate time to remission, and overcome single agent resistance are of considerable interest. We previously showed that the XPO1 inhibitor selinexor is proapoptotic in CLL cells and disrupts B-cell receptor signaling via BTK depletion. Herein we show the combination of selinexor and ibrutinib elicits a synergistic cytotoxic effect in primary CLL cells and increases overall survival compared with ibrutinib alone in a mouse model of CLL. Selinexor is effective in cells isolated from patients with prolonged lymphocytosis following ibrutinib therapy. Finally, selinexor is effective in ibrutinib-refractory mice and in a cell line harboring the BTK C481S mutation. This is the first report describing the combined activity of ibrutinib and selinexor in CLL, which represents a new treatment paradigm and warrants further evaluation in clinical trials of CLL patients including those with acquired ibrutinib resistance.

IMPORTANCE: The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is effective in patients with chronic lymphocytic leukemia (CLL). Reasons for discontinuing therapy with this drug and outcomes following discontinuation have not been evaluated outside of clinical trials with relatively short follow-up. OBJECTIVE: To determine features associated with discontinuation of ibrutinib therapy and outcomes. DESIGN, SETTING, AND PARTICIPANTS: A total of 308 patients participating in 4 sequential trials of ibrutinib at The Ohio State University Comprehensive Cancer Center were included. These clinical trials accrued patients included in this analysis from May 2010 until April 2014, and data were locked in June 2014. MAIN OUTCOMES AND MEASURES: Patients were evaluated for time to therapy discontinuation, reasons for discontinuation, and survival following discontinuation. For patients who discontinued therapy because of disease progression, targeted deep sequencing was performed in samples at baseline and time of relapse. RESULTS: With a median follow-up of 20 months, 232 patients remained on therapy, 31 had discontinued because of disease progression, and 45 had discontinued for other reasons. Disease progression includes Richter’s transformation (RT) or progressive CLL. Richter’s transformation appeared to occur early and CLL progressions later (cumulative incidence at 12 months, 4.5% [95% CI, 2.0%-7.0%] and 0.3% [95% CI, 0%-1.0%], respectively). Median survival following RT was 3.5 months (95% CI, 0.3-6.0 months) and 17.6 months (95% CI, 4.7 months–“not reached”) following CLL progression. Sequencing on peripheral blood from 8 patients with RT revealed 2 with mutations in BTK, and a lymph node sample showed no mutations in BTK or PLCG2. Deep sequencing on 11 patients with CLL progression revealed BTK or PLCG2 mutations in all. These mutations were not identified before treatment in any patient. CONCLUSIONS AND RELEVANCE: This single-institution experience with ibrutinib confirms it to be an effective therapy and identifies, for the first time, baseline factors associated with ibrutinib therapy discontinuation. Outcomes data show poor prognosis after discontinuation, especially for those patients with RT. Finally, sequencing data confirm initial reports associating mutations in BTK and PLCG2 with progression and clearly show that CLL progressions are associated with these mutations, while RT is likely not. TRIAL REGISTRATIONS: clinicaltrials.gov Identifiers:NCT01105247, NCT01217749, NCT01589302, and NCT01578707

Volume crime offences such as domestic burglary are commonly assessed for forensic opportunities by the first attending officer present at the scene. Conversely, less serious volume crime offences such as thefts from motor vehicles are very frequent and are routinely assessed for forensic opportunities by the victim talking to the police over the telephone. It is not clear whether this difference in attendance policy leads to differences in the types and quantity of forensic material recovered. The current study explored whether there was a benefit to evidence recovery for attended as opposed to non-attended assessments. Five hundred thefts from motor vehicles offences recorded by Northamptonshire Police (UK) between 14 January 2010 and 28 February 2011 were analysed; 250 were attended forensic assessments and 250 were non-attended assessments. Significant differences were found between the two scenarios, with attended assessments more likely to yield DNA, property and trace substance material. Conversely, fingerprints were more likely to be recovered at non-attended assessments. Despite the fruitful findings of the current study, future research would benefit from establishing the methods used by the first attending officer and forensic investigator when assessing and gathering evidence. Similarly, it is unclear whether these differences in forensic material are reflected in the identification of an offender and subsequently in the solving of the crime.

The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has outstanding activity in patients with chronic lymphocytic leukemia. Most patients experience lymphocytosis, representing lymphocyte egress from nodal compartments. This resolves within 8 months in the majority of patients, but a subgroup has lymphocytosis lasting >12 months. Here we report a detailed characterization of patients with persistent lymphocytosis during ibrutinib therapy. Signaling evaluation showed that while BTK is inhibited, downstream mediators of B-cell receptor (BCR) signaling are activated in persistent lymphocytes. These cells cannot be stimulated through the BCR and do not show evidence of target gene activation. Flow cytometry for κ and λ expression, IGHV sequencing, Zap-70 methylation, and targeted gene sequencing in these patients are identical at baseline and later time points, suggesting that persistent lymphocytes do not represent clonal evolution. In vitro treatment with targeted kinase inhibitors shows that they are not addicted to a single survival pathway. Finally, progression-free survival is not inferior for patients with prolonged lymphocytosis vs those with traditional responses. Thus, prolonged lymphocytosis is common following ibrutinib treatment, likely represents the persistence of a quiescent clone, and does not predict a subgroup of patients likely to relapse early.

Chronic lymphocytic leukemia (CLL) is characterized by constitutive activation of the B-cell receptor (BCR) signaling pathway, but variable responsiveness of the BCR to antigen ligation. Bruton’s tyrosine kinase (BTK) shows constitutive activity in CLL and is the target of irreversible inhibition by ibrutinib, an orally bioavailable kinase inhibitor that has shown outstanding activity in CLL. Early clinical results in CLL with other reversible and irreversible BTK inhibitors have been less promising, however, raising the question of whether BTK kinase activity is an important target of ibrutinib and also in CLL. To determine the role of BTK in CLL, we used patient samples and the Eμ-TCL1 (TCL1) transgenic mouse model of CLL, which results in spontaneous leukemia development. Inhibition of BTK in primary human CLL cells by small interfering RNA promotes apoptosis. Inhibition of BTK kinase activity through either targeted genetic inactivation or ibrutinib in the TCL1 mouse significantly delays the development of CLL, demonstrating that BTK is a critical kinase for CLL development and expansion and thus an important target of ibrutinib. Collectively, our data confirm the importance of kinase-functional BTK in CLL.

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.

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.

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-κB (NF-κB) 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, 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.

Drug development in human chronic lymphocytic leukemia (CLL) has been limited by lack of a suitable animal model to adequately assess pharmacologic properties relevant to clinical application. A recently described TCL-1 transgenic mouse develops a chronic B-cell CD5+ leukemia that might be useful for such studies. Following confirmation of the natural history of this leukemia in the transgenic mice, we demonstrated that the transformed murine lymphocytes express relevant therapeutic targets (Bcl-2, Mcl-1, AKT, PDK1, and DNMT1), wild-type p53 status, and in vitro sensitivity to therapeutic agents relevant to the treatment of human CLL. We then demonstrated the in vivo clinical activity of low-dose fludarabine in transgenic TCL-1 mice with active leukemia. These studies demonstrated both early reduction in blood-lymphocyte count and spleen size and prolongation of survival (P = .046) compared with control mice. Similar to human CLL, an emergence of resistance was noted with fludarabine treatment in vivo. Overall, these studies suggest that the TCL-1 transgenic leukemia mouse model has similar clinical and therapeutic response properties to human CLL and may therefore serve as a useful in vivo tool to screen new drugs for subsequent development in CLL.

Chronic lymphocytic leukemia (CLL) is an incurable adult leukemia characterized by disrupted apoptosis. OSU03012 is a bioavailable third-generation celecoxib derivative devoid of cyclooxygenase-2 inhibitory activity that potently induces apoptosis in prostate cancer cell lines and is being developed as an anticancer therapy in the National Cancer Institute (NCI) Rapid Access to Intervention Development (RAID) program. We assessed the ability of OSU03012 to induce apoptosis in primary CLL cells and the mechanism by which this occurs. The LC50 (lethal concentration 50%) of OSU03012 at 24 hours was 7.1 μM, and this decreased to 5.5 μM at 72 hours. Additionally, we have demonstrated that OSU03012 mediates apoptosis by activation of the intrinsic, mitochondrial pathway of apoptosis but also activates alternative cell death pathways that are caspase independent. The early activation of both caspase-dependent and -independent pathways of apoptosis is novel to OSU03012 and suggests it has great potential promise for the treatment of CLL. Moreover, unlike the great majority of therapeutic agents used to treat leukemia or other forms of cancer, OSU03012 induces cell death entirely independent of bcl-2 expression. Overall, these data provide justification for further preclinical development of OSU03012 as a potential therapeutic agent for CLL.

Woyach: Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding. Jones:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding. Andritsos:Hairy Cell Leukemia Foundation: Research Funding. Awan:Innate Pharma: Research Funding; Pharmacyclics: Consultancy; Novartis Oncology: Consultancy. Lozanski:Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Boehringer Ingelheim: Research Funding; Genentech: Research Funding.

Eathiraj: ArQule, Inc: Employment. Abbadessa:ArQule, Inc: Employment. Schwartz:ArQule, Inc: Employment. Woyach:Acerta: Research Funding; Morphosys: Research Funding; Karyopharm: Research Funding.

Woyach: Acerta: Research Funding; Karyopharm: Research Funding; Morphosys: Research Funding.

Woyach: Karyopharm: Research Funding; Morphosys: Research Funding; Acerta: Research Funding.

Byrd: Acerta Pharma BV: Research Funding.

Chang: Pharmacyclics, Inc.: Employment. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Jaglowski: Pharmacyclics: Research Funding. Chang:Pharmacyclics, Inc.: Employment. Maddocks:Pharmacyclics: Research Funding. Buggy:Pharmacyclics: Employment, Equity Ownership. Byrd:Pharmacyclics: Research Funding.

Jones: Glaxo Smith-Kline: Consultancy; Abbott: Research Funding. Bannerji:Merck & Co: Employment, Equity Ownership.

Small: Schering Plough: Employment. Statkevich:Schering Plough: Employment. Bannerji:Schering-Plough: Employment.

Lenalinomide is an immunomodulatory agent with activity in hematologic malignancies including multiple myeloma and myelodysplastic syndrome. Recent phase II studies have demonstrated that lenalidomide has significant activity in chronic lymphocytic leukemia (CLL). Patients included in this report including a single patient treated off study based on available data for cytoreduction for planned allogeneic stem cell transplantation. Three additional patients with relapsed CLL were enrolled on a IRB approved phase I study evaluating the optimal initial dose in CLL. All patients were treated at 25 mg/day for 21 days with a 7 day off period. Because of previous reports of tumor lysis syndrome and tumor flare during initiation of therapy, patients were initially monitored daily in the outpatient setting with physical examinations and laboratory evaluation. All patients received prophylactic corticosteroids consisting of dexamethasone 12 mg orally days 1–7 followed by 4 mg orally days 7–14 except patient #1, who received prednisone 20 mg daily. Patient specific histories include: Patient #1 had bulky lymphadenopathy and a WBC count of 197,000 at the time of initiation of therapy. On day 3 of therapy he omitted his steroid prophylaxis and rapidly developed a syndrome of worsening lymphocytosis, lymph node enlargement, acute renal failure with laboratory evidence of tumor lysis syndrome, and despite mechanical ventilation died of hypoxemic respiratory failure on day 10 of therapy. Patient #2 initially did well, however on day 25 of course 1 presented to an outside facility with gram negative sepsis in the setting of neutropenia. He recovered fully, however soon after this episode developed disease progression and was taken off study. Patient #3 was asymptomatic until day 8 of course 1, when he developed symptoms of tumor flare during steroid taper with severe abdominal and back pain, skin rash, and low grade fevers. His symptoms resolved with cessation of therapy. He subsequently underwent serial dose reductions but continued to develop signs and symptoms of tumor flare requiring steroid therapy and narcotic pain medication. Patient #4 initiated therapy at 25 mg/day and did well during his steroid taper, however on day 25 while off therapy presented for evaluation of shortness of breath and airway occlusion with recumbency. He was found to have dramatic enlargement of cervical and supraclavicular lymph nodes as well as new tonsillar hypertrophy obstructing his airway; tonsillectomy demonstrated CLL without transformation in the tonsillar tissues. He received steroids with improvement and lenalidomide was discontinued. Lenalidomide administered at 25 mg orally daily is associated with unacceptable toxicity due to myelosuppression, tumor lysis syndrome, and tumor flare. Tumor flare may be indistinguishable from disease progression and may not respond to withdrawal of therapy and steroid administration. Future studies should be designed to initiate therapy at a low dose with gradual dose escalation, with close monitoring for complications. Given the uncertainty of safe dose and schedule of lenalidomide, use of this therapy off an IRB approved study that includes close patient monitoring for tumor flare should not be considered.

Background: New therapies for relapsed CLL pts with poor-risk cytogenetic features, such as del(11q22), del(17p13) or a complex karyotype, are needed. Alemtuzumab (Campath-1H) is effective in this pt population but has infectious toxicity and is ineffective as a single agent in bulky nodal disease. Flavopiridol induces p53-independent apoptosis in CLL cells in vitro. We previously demonstrated the activity of flavopiridol in genetically high-risk CLL in a phase I study using a novel, pharmacokinetically (PK) derived dosing schedule, and we are currently pursuing a larger phase II trial of this drug. We present updated results of flavopiridol’s activity in high-risk pts in both studies. Study Design and Treatment: Pts (n=89) with symptomatic, relapsed CLL who failed (or could not receive) fludarabine received flavopiridol by 30-min IVB followed by 4-hr CIVI weekly for 4 doses, every 6 weeks for up to 6 cycles. Pts received 30 mg/m2 IVB + 30 mg/m2 CIVI (n=20), 40 mg/m2 IVB + 40 mg/m2 CIVI (n=3), 30 mg/m2 IVB + 30 mg/m2 CIVI for the first 4 doses and 30 mg/m2 IVB + 50 mg/m2 CIVI for all subsequent doses (n=14) or 30 mg/m2 IVB + 30 mg/m2 CIVI for the first dose and 30 mg/m2 IVB + 50 mg/m2 CIVI for all subsequent doses (n=44). Eight pts who experienced severe tumor lysis syndrome did not undergo dose escalation as planned. A complex karyotype was defined as ≥ 3 unrelated abnormalities, and FISH for TP53 [del(17p13)] and ATM [del(11q22)] determined loss of those genes (regions). Response Assessment: Median age was 61 years (range, 38–84), with 21 pts ≥ 70 years of age. Median number of prior therapies was 5 (range, 1–14); 87 pts had failed fludarabine therapy. Pts had bulky Rai stage I/II (n=18), III (19) or IV (n=52) disease. Pts received a median of 3 cycles (range, 0.25–6), 11 pts completed all 6 cycles, and 2 continue to receive therapy. All 89 pts were evaluated for response, including 6 pts who received only one dose due to grade 4–5 tumor lysis or other complications. Forty-one pts responded (46%) by NCI Working Group response criteria; 39 pts achieved a partial response (PR, 44%), and 2 pts with trisomy 12 achieved a complete response (CR, 2%). Fourteen of 29 pts with del(17p13) responded (48%), 22 of 37 pts with del(11q22) responded (59%), and 20 of 47 pts with a complex karyotype responded (43%). Thirty-three of 70 pts with bulky adenopathy >5 cm responded (47%). Progression free survival data will be updated at ASH. Conclusions: Flavopiridol demonstrates significant clinical activity as a single agent in heavily pretreated, relapsed CLL pts with bulky adenopathy and poor-risk cytogenetic features such as del(17p13), del(11q22) and complex karytoypes. Further studies of this drug in CLL and other hematologic malignancies are ongoing. All Patients Loss TP53 Loss ATM Complex Karyotype Bulky nodes > 5 cm Patients 89 29 37 47 70 Partial Response 39 14 22 20 32 Complete response 2 0 0 0 1 No response 48 15 15 27 37

Background: Relapsed CLL patients (pts) with del(17p13) and other high-risk genetic features respond poorly to most standard therapies. Flavopiridol (alvocidib) induces p53-independent apoptosis of CLL cells in vitro. We previously conducted a phase I study of flavopiridol using a pharmacokinetically (PK) derived dosing schedule of 30-min IV bolus (IVB) followed by 4-hr continuous IV infusion (CIVI). Clinical activity (response rate 45%) was seen in high-risk pts, but several pts required hemodialysis for severe tumor lysis syndrome (TLS) and hyperkalemia. Study Design and Treatment: We report preliminary results of an ongoing phase II study of flavopiridol in relapsed CLL. Pts with symptomatic, relapsed CLL who have failed (or could not receive) fludarabine and have WBC < 200 × 109/L are eligible. Pts receive flavopiridol by 30-min IVB followed by 4-hr CIVI weekly for 4 doses, every 6 weeks for up to 6 cycles. Pts receive 30 mg/m2 IVB + 30 mg/m2 CIVI for dose 1, and pts receive 30 mg/m2 IVB + 50 mg/m2 CIVI with the second and all subsequent doses if severe TLS is not observed. Results: We report results of the first 31 pts (19 male). Median age was 65 years (range, 41–82), with 9 pts ≥ 70 years of age. Median number of prior therapies was 6 (range, 1–11), and 30 pts had failed fludarabine. Pts had bulky Rai stage I/II (n=5), III (n=5) or IV (n=21) disease, and 27 pts had bulky lymphadenopathy ≥ 5 cm. Therapy was well tolerated. No patients required hemodialysis, and toxicity was otherwise similar to the phase I study. Cytokine release syndrome related to interleukin (IL)-6 was observed in a majority of pts, and symptoms responded to dexamethasone. Pts received a median of 3 cycles (range, 0.25–6). Two pts completed all 6 cycles, and 2 pts continue to receive therapy. The most common reasons for early discontinuation of therapy were failure to respond (n=11) and patient choice (n=6). Fifteen of 31 pts responded (48%) by NCI Working Group criteria; 13 pts achieved a partial response (PR), and 2 pts attained CR. One CR pt achieved a flow negative bone marrow (BM), and the other CR pt had < 1% residual CLL in BM by flow cytometry. Five of 9 pts with del(17p13) responded (56%), 5 of 15 pts with del(11q22) responded (33%), and 7 of 18 pts with a complex karyotype responded (39%). Follow-up remains short, but progression-free survival will be updated. Conclusions: This study confirms the significant clinical activity of flavopiridol in heavily pretreated, relapsed CLL pts with bulky adenopathy and poor-risk cytogenetic features. Furthermore, 2 pts achieved CR, including 1 pt with flow-negative BM. Limiting eligibility to WBC < 200 × 109/L improved safety, and no pts required dialysis. However, cytokine release syndrome related to IL-6 was common and caused pts to elect to end therapy. Therefore, this study has been amended to give prophylactic dexamethasone, and the schedule has been shortened with the use of prophylactic pegfilgrastim, in order to improve tolerability. Accrual to the amended study is ongoing.

The heat shock protein Hsp90 functions to stabilize important cell survival- and proliferation-related kinases such as AKT, IKK, c-Src, Raf-1, and cdk9. Cancer cells have activated Hsp90 as compared to normal cells, making this a relevant therapeutic target to eliminate these kinases. The semi-synthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) binds to and inhibits the activity of Hsp90, and previous work demonstrated the ability of 17-AAG to deplete AKT in several cancer types in vitro. A newer geldanamycin derivative, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) has improved pharmacological properties including solubility and oral bioavailability, and was shown to be more effective than 17-AAG in melanoma and pancreatic carcinoma mouse xenograft mouse models. We therefore examined the effects of 17-AAG and DMAG against CD19-positive tumor cells from CLL patients. Cell viability was examined by the MTT assay, and AKT and IKK protein expression was examined by immunoblot analysis. In samples from seven CLL patients, 1.0uM DMAG resulted in 31.5% viability (95% CI: 13.1–50.0%), compared to 61.5% viability (95% CI: 45.0–78.0%) using the same concentration of 17-AAG. In four CLL patient samples treated with 1.0uM DMAG for 24 hours, AKT was decreased an average of 72.5% (95% CI: 57.7–87.3%) relative to the untreated controls. This is in comparison to 1.0uM 17-AAG, which caused a 52.7% decrease in AKT (95%CI: 39.7–65.6%). IKK protein was also decreased at similar levels in all patient samples examined. This data indicates that in CLL cells, DMAG has superior activity both in cytotoxicity and in reduction of relevant Hsp90 client proteins. 17-AAG is currently undergoing Phase I clinical testing in CLL, and DMAG is completing phase I clinical development in solid tumor malignancies. Overall, our data and that of others support clinical development of DMAG in CLL, based on the improved pharmacologic properties, enhanced efficacy relative to 17-AAG, and expected down-regulation of target proteins. In addition, our in vitro observations support using measurement of protein down-regulation of AKT and IKK as pharmacodynamic biomarkers of activity in patients undergoing therapy with these agents.

We have noted impressive activity of the cyclin-dependent kinase inhibitor flavopiridol in advanced-stage CLL patients, including those with a deletion of chromosome 17p13. Using a novel, effective schedule of flavopiridol, substantial and sometimes dramatic evidence of tumor cell death is observed as early as 4–6 hours. This is accompanied by hyperkalemia, hyperphosphatemia, hypocalcemia, and dramatic elevation in LDH consistent with acute tumor lysis syndrome. Studies by several groups including our own have demonstrated that Mcl-1 protein and mRNA is down-regulated with flavopiridol. Mcl-1 is an important protein that contributes to mitochondrial membrane stability. We have therefore sought to determine the role of mitochondria disruption in the mechanism of action of flavopiridol. By flow cytometry using the voltage-sensitive dye JC-1, loss of mitochondrial membrane potential was detected in flavopiridol-treated whole blood as early as five hours, prior to the onset of annexin-V or propidium iodide staining. This is in contrast to in vitro studies using human serum, in which mitochondrial depolarization and annexin-V staining occurred simultaneously. In isolated CLL cells treated with flavopiridol in vitro, loss of mitochondrial membrane potential was not affected by inhibitors of caspases 8 or 9 or by the broad caspase inhibitor Z-VAD-fmk, although apoptosis was effectively blocked by Z-VAD-fmk and caspase-8 inhibitor, and to a lesser extent, caspase-9 inhibitor. Flavopiridol was also able to effectively induce apoptosis and mitochondrial membrane depolarization in Jurkat cell lines deficient in caspase-8 or its adapter protein FADD. Additionally, lymphoid cells overexpressing Bcl-2 are resistant to flavopiridol-mediated apoptosis relative to the vector control. This suggests there is not direct binding of flavopiridol or its metabolites to APAF-1 (cytosolic adapter protein) and apoptosome assembly, as this process is insensitive to Bcl-2 family proteins. Further mechanistic studies were undertaken using isolated liver mitochondria. While the electron transport system was not uncoupled in this system, potential mechanisms of mitochondrial injury in leukemic cells from CLL patients are currently under exploration. Taken together, these observations suggest that mitochondrial perturbation contributes significantly to the death process induced by flavopiridol. Further studies to identify the mechanism of mitochondrial perturbation will be essential to understanding flavopiridol’s mechanism of action and for predicting patients at risk for acute tumor lysis syndrome. (Support for this work was provided by the Samuel Waxman Foundation and the Leukemia & Lymphoma Society.)

A TCL-1 transgenic mouse model of CLL was recently described that develops a slowly proliferating CD19+/CD5+/IgM+ clonal leukemia at 10–13 months of age, characterized by lymphocytosis, splenomegaly, and lymphadenopathy. To determine if this model has therapeutic properties similar to human CLL, we performed in vitro cytotoxicity assays using splenic B-cells isolated from leukemic TCL-1 mice. Results indicated that fludarabine, a common treatment for CLL, as well as experimental agents, 17-AAG, valproic acid, flavopiridol, and a novel kinase inhibitor OSU03012, each have activity against the mouse TCL-1-mediated leukemia at concentrations similar to that observed in human CLL cells in vitro. Subsequent in vivo studies in TCL-1 transgenic mice with evidence of leukemia were performed with low dose fludarabine (34 mg/kg IP, day 1–5, every month) versus saline control (n=10 per group) and were treated for three cycles. Mice receiving fludarabine had a notable decline in blood lymphocyte count. Furthermore, the median survival time for the fludarabine treated mice was 61 days, as compared to 31 days for the control mice (p=0.046). However, as seen in human CLL, treated mice became resistant to fludarabine over time as determined by lymphocyte counts. Thus, our preliminary data suggest the TCL-1 transgenic mouse model of CLL has a similar therapeutic response pattern to human CLL. We next assessed the ability of a novel PDK1/AKT inhibitor OSU03012 to mediate in vivo activity in this model. This agent previously has been shown to mediate activity against both CLL cells and TCL-1 leukemia cells in vitro through a caspase and bcl-2 independent pathway. Surprisingly, OSU03012 demonstrated no early or late in vivo activity at a dose (200 mg/kg) previously demonstrated to inhibit prostate xenograft tumor growth in vivo. The lack of efficacy with this compound in the TCL-1 transgenic mouse prompted us to examine in vivo properties such as protein binding, non-linear absorption of drug that could prevent attaining the concentration of free drug necessary to mediate apoptosis, or stromal cell contact that could promote cellular resistance to OSU03012. As measured using a liquid chromatography/tandem mass spectrometry assay, plasma levels of OSU03012 reached peak levels of 7.5–10μM in the treated mice. Interestingly, very high protein binding of OSU03012 to murine plasma (98–99%) was detected. This finding was further corroborated by parallel in vitro cytotoxicity studies in fetal bovine serum and mouse serum, in which the IC50 concentrations were 3.558μM and 14.15μM, respectively. These studies indicated that plasma levels of OSU03012 sufficient to mediate cytotoxicity against TCL-1 leukemia cells were not achieved in vivo. A second therapeutic study using higher doses of OSU03012 is underway to attain these higher concentrations to confirm in vivo activity of OSU03012. Preclinical studies of other highly effective agents (e.g., Flavopiridol) have recently demonstrated the importance of defining differences in plasma protein binding and schedule of administration of novel agents. It is important to consider the impact of plasma protein binding in designing early phase I studies in patients. Hopefully, this murine model will facilitate future preclinical studies to optimize the schedule and dose of drug administration to confirm the in vivo activity of promising new agents, including OSU03012.