Varsha Gandhi

The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo

B-cell receptor (BCR) signaling is a critical pathway in the pathogenesis of several B-cell malignancies, including chronic lymphocytic leukemia (CLL), and can be targeted by inhibitors of BCR-associated kinases, such as Bruton tyrosine kinase (Btk). PCI-32765, a selective, irreversible Btk inhibitor, is a novel, molecularly targeted agent for patients with B-cell malignancies, and is particularly active in patients with CLL. In this study, we analyzed the mechanism of action of PCI-32765 in CLL, using in vitro and in vivo models, and performed correlative studies on specimens from patients receiving therapy with PCI-32765. PCI-32765 significantly inhibited CLL cell survival, DNA synthesis, and migration in response to tissue homing chemokines (CXCL12, CXCL13). PCI-32765 also down-regulated secretion of BCR-dependent chemokines (CCL3, CCL4) by the CLL cells, both in vitro and in vivo. In an adoptive transfer TCL1 mouse model of CLL, PCI-32765 affected disease progression. In this model, PCI-32765 caused a transient early lymphocytosis, and profoundly inhibited CLL progression, as assessed by weight, development, and extent of hepatospenomegaly, and survival. Our data demonstrate that PCI-32765 effectively inhibits CLL cell migration and survival, possibly explaining some of the characteristic clinical activity of this new targeted agent.

An Epithelial–Mesenchymal Transition Gene Signature Predicts Resistance to EGFR and PI3K Inhibitors and Identifies Axl as a Therapeutic Target for Overcoming EGFR Inhibitor Resistance

Purpose: Epithelial–mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non–small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study. Experimental Design: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified. Results: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies. Conclusion: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype. Clin Cancer Res; 19(1); 279–90. ©2012 AACR.

Fludarabine potentiates metabolism of cytarabine in patients with acute myelogenous leukemia during therapy.

PURPOSE A protocol was designed to test the hypothesis that fludarabine infusion before arabinosylcytosine (cytarabine [ara-C]) would increase the accumulation of the active metabolite ara-C triphosphate (ara-CTP) in acute myelogenous leukemia (AML) blasts during therapy. PATIENTS AND METHODS Patients (n = 5) received 1 g/m2 of ara-C infused intravenously (IV) for 2 hours, followed at 20 hours by 30 mg/m2 of fludarabine for 30 minutes. At 24 hours, another identical dose of ara-C was infused. To determine the optimal duration of ara-C infusion following fludarabine, five additional patients were treated on an amended protocol in which the ara-C infusion was extended to 3 g/m2 infused over 6 hours. RESULTS Comparison of ara-CTP pharmacokinetics in circulating AML cells demonstrated that the area under the curve (AUC) of ara-CTP increased significantly (median, 1.8-fold; range, 1.6 to 2.4; P = .004) after fludarabine infusion. Neither the median plasma ara-C concentrations, the levels of its deamination product arabinosyluracil, nor the rate of ara-CTP elimination from circulating blasts was affected by fludarabine infusion. However, the rate of ara-CTP accumulation by AML cells was increased by a median of 2.0-fold (range, 1.8 to 2.2; P = .001) after fludarabine; the peak occurred within 1 hour of the end of the infusion. In vitro incubation of these cells with arabinosyl-2-fluoroadenine (F-ara-A) before ara-C also produced a median 1.7-fold increase in the ara-CTP accumulation rate. Pharmacology studies in patients receiving 6-hour infusions of ara-C demonstrated that the rate of ara-CTP accumulation was potentiated beyond 2 hours, but not for 6 hours. CONCLUSION Infusion of fludarabine before ara-C augments the rate of ara-CTP synthesis in circulating AML blasts during therapy. Evaluation of 6-hour ara-C infusions demonstrated that potentiation of ara-CTP synthesis is maximal up to 4 hours in most patients; this pharmacologically optimized regimen should be considered for combination with other antileukemia drugs.

Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL-MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL-MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell-cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC-CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.

Preclinical characteristics of gemcitabine

Gemcitabine (2I, 2I-difluorodeoxycytidine, dFdC) is a nucleoside analogue of deoxycytidine in which two fluorine atoms have been inserted into the deoxyribofuranosyl ring. Once inside the cell gemcitabine is rapidly phosphorylated by deoxycytidine kinase, the rate-limiting enzyme for the formation of the active metabolites gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP). Gemcitabine diphosphate inhibits ribonucleotide reductase, which is responsible for producing the deoxynucleotides required for DNA synthesis and repair. The subsequent decrease in cellular deoxynucleotides (particularly dCTP) favours gemcitabine triphosphate in its competition with dCTP for incorporation into DNA. Reduction in cellular dCTP is an important self-potentiating mechanism resulting in increased gemcitabine nucleotide incorporation into DNA. Other self-potentiating mechanisms of gemcitabine include increased formation of active gemcitabine di- and triphosphates, and decreased elimination of gemcitabine nucleotides. After gemcitabine nucleotide is incorporated on the end of the elongating DNA strand, one more deoxynucleotide is added, and thereafter the DNA polymerases are unable to proceed. This action, termed “masked chain termination”, appears to lock the drug into DNA because proof-reading exonucleases are unable to remove gemcitabine nucleotide from this penultimate position. Incorporation of gemcitabine triphosphate into DNA is strongly correlated with the inhibition of further DNA synthesis. Compared with ara-C, gemcitabine serves as a better transport substrate, is phosphorylated more efficiently, and is eliminated more slowly. These differences, together with self-potentiation, masked chain termination and the inhibition of ribonucleotide reductase, which are not seen with ara-C, may explain why gemcitabine is, and ara-C is not, active in solid tumours. This unique combination of metabolic properties and mechanistic characteristics suggests that gemcitabine is likely to be synergistic with other drugs that damage DNA, and also with other modalities such as radiation.

Phase II Clinical Investigation of Gemcitabine in Advanced Soft Tissue Sarcomas and Window Evaluation of Dose Rate on Gemcitabine Triphosphate Accumulation

PURPOSE To evaluate the efficacy, toxicity, and optimal dose rate of gemcitabine in adult patients with advanced soft tissue sarcomas (STS) by comparing levels of gemcitabine triphosphate (GTP) in peripheral-blood mononuclear cells (PBMCs) of patients receiving two different dose rates. PATIENTS AND METHODS Fifty-six assessable patients with STS (17 gastrointestinal [GI] leiomyosarcomas and 39 other histologies) were treated on a two-arm phase II study. Gemcitabine was given at 1 g/m2 as a 30-minute infusion weekly for up to 7 weeks followed by 1 week of rest and reassessment of tumor. Subsequent cycles were given at 1 g/m2 weekly for 3 weeks followed by 1 week of rest. Nine patients underwent cellular pharmacologic studies at two different dose rates (1 g/m2 over a standard 30-minute infusion on week 1 and over pharmacologically based infusion of 150 minutes on week 2) to evaluate GTP levels in PBMCs. RESULTS Seven partial responses were noted among 39 patients, for an overall response rate of 18% (95% confidence interval, 7% to 29%). Median duration of response was 3.5 months (range, 2 to 13 months). Four of 10 patients with non-GI leiomyosarcomas achieved a partial response. No objective responses were noted in 17 patients with GI leiomyosarcomas. One patient had a mixed response. Median time to progression for all patients (both arms) was 3 months; median survival was 13.9 months. Treatment was generally well tolerated. Comparison of cellular pharmacology demonstrated a significant 1.4-fold increase in the concentration of GTP with the 150-minute infusion. CONCLUSION Given the limited therapeutic armamentarium for STS, the activity of gemcitabine is encouraging. Its potential for combination therapy in the salvage setting should be studied with pharmacologically guided fixed dose-rate infusion.

B-cell antigen receptor signaling enhances chronic lymphocytic leukemia cell migration and survival: specific targeting with a novel spleen tyrosine kinase inhibitor, R406.

Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.

Cellular and clinical pharmacology of fludarabine

In the past decade, fludarabine has had a major impact in increasing the effectiveness of treatment of patients with indolent B-cell malignancies. This has come about in a variety of clinical circumstances, including use of fludarabine alone as well as in combinations with DNA-damaging agents or membrane-targeted antibodies. Other strategies have used fludarabine to reduce immunological function, thus facilitating non-myeloablative stem cell transplants.Fludarabine is a prodrug that is converted to the free nucleoside 9-β-D-arabinosyl-2-fluoroadenine (F-ara-A) which enters cells and accumulates mainly as the 5′-triphosphate, F-ara-ATP. The rate-limiting step in the formation of triphosphate is conversion of F-ara-A to its monophosphate, which is catalyzed by deoxycytidine kinase. Although F-ara-A is not a good substrate for this enzyme, the high specific activity of this protein results in efficient phosphorylation of F-ara-A in certain tissues. F-ara-ATP has multiple mechanisms of action, which are mostly directed toward DNA. These include inhibition of ribonucleotide reductase, incorporation into DNA resulting in repression of further DNA polymerisation, and inhibition of DNA ligase and DNA primase. Collectively these actions affect DNA synthesis, which is the major mechanism of F-ara-A-induced cytotoxicity Secondarily, incorporation into RNA and inhibition of transcription has been shown in cell lines.With the standard dose of fludarabine (25 to 30 mg/m2/day given over 30 minutes for 5 days), plasma concentrations of about 3 μmol/L F-ara-A are achieved at the end of each infusion. Serial sampling of leukaemia cells from patients receiving these standard doses of fludarabine has demonstrated that the peak concentrations of F-ara-ATP are achieved 4 hours after start of fludarabine infusion. Although there is heterogeneity among individuals with respect to rate of F-ara-ATP accumulation, the peak concentrations are generally proportional to the dose of the drug. Knowledge of the plasma pharmacokinetics of its principal nucleoside metabolite F-ara-A, and the cellular pharmacology of the proximal active metabolite, F-ara-ATP, has provided some understanding of the activity of fludarabine when used as a single agent. Preclinical studies directed toward learning the mechanisms of action of this agent have formed the basis for several mechanism-based strategies for its combination and scheduling with other agents.As a single agent fludarabine has been effective for the indolent leukaemias. Biochemical modulation strategies resulted in enhanced accumulation of cytarabine triphosphate and led to the use of fludarabine for the treatment of acute leukaemias. Combination of fludarabine with DNA damaging agents to inhibit DNA repair processes has been highly effective for indolent leukaemias and lymphomas. The current review brings together knowledge of the mechanisms of fludarabine, the state of understanding of the plasma pharmacokinetics, and cellular pharmacodynamics of fludarabine nucleotides. This may be useful in the design of future therapeutic approaches.

Phase I Clinical and Pharmacology Study of Clofarabine in Patients With Solid and Hematologic Cancers

PURPOSE To define the maximum-tolerated doses (MTDs) and dose-limiting toxicities (DLTs) of clofarabine, given as a 1-hour infusion daily for 5 days, in patients with solid tumors and with acute leukemia. PATIENTS AND METHODS The initial part of the study defined the MTD and DLT in solid tumors. The second part of the study defined the MTD and DLT in acute leukemia. RESULTS The starting dose of clofarabine (15 mg/m(2)) was myelosuppressive, requiring several dose de-escalations to 2 mg/m(2), the dose suggested for phase II studies in solid tumors. Dose escalation in acute leukemia started at 7.5 mg/m(2), with several escalations to 55 mg/m(2). The DLT was reversible hepatotoxicity at 55 mg/m(2). The recommended dose for acute leukemia phase II studies was 40 mg/m(2). Among 32 treated patients with acute leukemia, two achieved a complete response and three had a marrow complete response without platelet recovery (hematologic improvement), for an overall response rate of 16%. At 40 mg/m(2), the median plasma clofarabine level was 1.5 micro mol/L (range, 0.42 to 3.2 micro mol/L; n = 7). Cellular and plasma pharmacokinetic studies suggested dose proportionality but showed a wide variation in intracellular concentrations of clofarabine triphosphate. CONCLUSION This phase I study defined the following two MTDs for clofarabine given as a 1-hour infusion daily for 5 days: 2 mg/m(2) for solid tumors, the DLT being myelosuppression; and 40 mg/m(2) for acute leukemia, the DLT being hepatotoxicity. Encouraging activity was observed in acute leukemia.

Fludarabine and Arabinosylcytosine Therapy of Refractory and Relapsed Acute Myelogenous Leukemia

There is a strong association between ability of leukemia blasts to accumulate ara-CTP, the active metabolite of ara-C, and response to ara-C in patients with relapsed or refractory AML. Ara-C dose rates in excess of 0.5 g/m2/h do not produce further ara-CTP formation. In contrast, when given 4 h prior to ara-C at this dose rate, fludarabine, at doses that are free of neurotoxicity in CLL, enhances ara-CTP accumulation. This led us to administer fludarabine and ara-C to 59 patients with AML in relapse or unresponsive to initial therapy. Fludarabine was given at 30 mg/m2 once daily for 5 doses and ara-C at 0.5 g/m2/h for 2-6 h daily for 6 doses. Doses of fludarabine preceded those of ara-C by 4 h. Results with fludarabine and ara-C (FA) were compared with those of patients treated at M.D. Anderson with high-dose ara-C (HDAC) or intermediate-dose ara-C (IDAC). The complete remission rate with FA was 21/59 (36%) and the actuarial median CR duration 39 weeks. FA produced significantly higher remission rates than HDAC or IDAC in patients with initial remissions > 1 yr (14/20 vs 9/23 vs 6/18, p < 0.05). Response rates were similar for all three treatments in patients with initial remissions < 1 yr or with primary refractory disease. The regimen was well tolerated; one patient developed peripheral neuropathy. This low level of toxicity encourages combination with other antileukemia agents.