Kevin L. Peterson

Phase I and Pharmacologic Trial of Cytosine Arabinoside with the Selective Checkpoint 1 Inhibitor Sch 900776 in Refractory Acute Leukemias

Purpose: Incorporation of cytarabine into DNA activates checkpoint kinase 1 (Chk1), which stabilizes stalled replication forks, induces S-phase slowing, and diminishes cytarabine cytotoxicity. The selective Chk1 inhibitor SCH 900776 abrogates cytarabine-induced S-phase arrest and enhances cytarabine cytotoxicity in acute leukemia cell lines and leukemic blasts in vitro. To extend these findings to the clinical setting, we have conducted a phase I study of cytarabine and SCH 900776. Experimental Design: Twenty-four adults with relapsed and refractory acute leukemias received timed sequential, continuous infusion cytarabine 2 g/m2 over 72 hours (667 mg/m2/24 hours) beginning on day 1 and again on day 10. SCH 900776 was administered as a 15- to 30-minute infusion on days 2, 3, 11, and 12. The starting dose of SCH 900776 was 10 mg/m2/dose. Results: Dose-limiting toxicities consisting of corrected QT interval prolongation and grade 3 palmar-plantar erythrodysesthesia occurred at 140 mg flat dosing (dose level 5, equivalent to 80 mg/m2). Complete remissions occurred in 8 of 24 (33%) patients, with 7 of 8 at 40 mg/m2 or higher. SCH 900776 did not accumulate at any dose level. Marrow blasts obtained pretreatment and during therapy showed increased phosphorylation of H2Ax after SCH 900776 beginning at 40 mg/m2, consistent with unrepaired DNA damage. Conclusions: These data support a randomized phase II trial of cytarabine +/− SCH 900776 at a recommended flat dose of 100 mg (equivalent to 56 mg/m2) for adults with poor-risk leukemias. The trial (SP P05247) was registered at www.clinicaltrials.gov as NCT00907517. Clin Cancer Res; 18(24); 6723–31. ©2012 AACR.

Dual mTORC1/mTORC2 inhibition diminishes Akt activation and induces Puma-dependent apoptosis in lymphoid malignancies.

The mammalian target of rapamycin (mTOR) plays crucial roles in proliferative and antiapoptotic signaling in lymphoid malignancies. Rapamycin analogs, which are allosteric mTOR complex 1 (mTORC1) inhibitors, are active in mantle cell lymphoma and other lymphoid neoplasms, but responses are usually partial and short-lived. In the present study we compared the effects of rapamycin with the dual mTORC1/mTORC2 inhibitor OSI-027 in cell lines and clinical samples representing divers lymphoid malignancies. In contrast to rapamycin, OSI-027 markedly diminished proliferation and induced apoptosis in a variety of lymphoid cell lines and clinical samples, including specimens of B-cell acute lymphocytic leukemia (ALL), mantle cell lymphoma, marginal zone lymphoma and Sezary syndrome. Additional analysis demonstrated that OSI-027-induced apoptosis depended on transcriptional activation of the PUMA and BIM genes. Overexpression of Bcl-2, which neutralizes Puma and Bim, or loss of procaspase 9 diminished OSI-027-induced apoptosis in vitro. Moreover, OSI-027 inhibited phosphorylation of mTORC1 and mTORC2 substrates, up-regulated Puma, and induced regressions in Jeko xenografts. Collectively, these results not only identify a pathway that is critical for the cytotoxicity of dual mTORC1/mTORC2 inhibitors, but also suggest that simultaneously targeting mTORC1 and mTORC2 might be an effective anti-lymphoma strategy in vivo.

Heat Shock Protein 90 Inhibition Depletes LATS1 and LATS2, Two Regulators of the Mammalian Hippo Tumor Suppressor Pathway

Heat shock protein 90 (HSP90), which regulates the functions of multiple oncogenic signaling pathways, has emerged as a novel anticancer therapeutic target, and multiple small-molecule HSP90 inhibitors are now in clinical trials. Although the effects of HSP90 inhibitors on oncogenic signaling pathways have been extensively studied, the effects of these agents on tumor suppressor signaling pathways are currently unknown. Here, we have examined how HSP90 inhibitors affect LATS1 and the related protein LATS2, two kinases that relay antiproliferative signals in the Hippo tumor suppressor pathway. Both LATS1 and LATS2 were depleted from cells treated with the HSP90 inhibitors 17-allylamino-17-demethoxygeldanamycin (17-AAG), radicicol, and PU-H71. Moreover, these kinases interacted with HSP90, and LATS1 isolated from 17-AAG-treated cells had reduced catalytic activity, thus showing that the kinase is a bona fide HSP90 client. Importantly, LATS1 signaling was disrupted by 17-AAG in tumor cell lines in vitro and clinical ovarian cancers in vivo as shown by reduced levels of LATS1 and decreased phosphorylation of the LATS substrate YAP, an oncoprotein transcriptional coactivator that regulates genes involved in cell and tissue growth, including the CTGF gene. Consistent with the reduced YAP phosphorylation, there were increased levels of CTGF, a secreted protein that is implicated in tumor proliferation, metastasis, and angiogenesis. Taken together, these results identify LATS1 and LATS2 as novel HSP90 clients and show that HSP90 inhibitors can disrupt the LATS tumor suppressor pathway in human cancer cells.

CHK1 and WEE1 inhibition combine synergistically to enhance therapeutic efficacy in acute myeloid leukemia ex vivo.

Novel combinations targeting new molecular vulnerabilities are needed to improve the outcome of patients with acute myeloid leukemia. We recently identified WEE1 kinase as a novel target in leukemias. To identify genes that are synthetically lethal with WEE1 inhibition, we performed a short interfering RNA screen directed against cell cycle and DNA repair genes during concurrent treatment with the WEE1 inhibitor MK1775. CHK1 and ATR, genes encoding two replication checkpoint kinases, were among the genes whose silencing enhanced the effects of WEE1 inhibition most, whereas CDK2 short interfering RNA antagonized MK1775 effects. Building on this observation, we examined the impact of combining MK1775 with selective small molecule inhibitors of CHK1, ATR and cyclin-dependent kinases. The CHK1 inhibitor MK8776 sensitized acute myeloid leukemia cell lines and primary leukemia specimens to MK1775 ex vivo, whereas smaller effects were observed with the MK1775/MK8776 combination in normal myeloid progenitors. The ATR inhibitor VE-821 likewise enhanced the antiproliferative effects of MK1775, whereas the cyclin-dependent kinase inhibitor roscovitine antagonized MK1775. Further studies showed that MK8776 enhanced MK1775-mediated activation of the ATR/CHK1 pathway in acute leukemia cell lines and ex vivo. These results indicate that combined cell cycle checkpoint interference with MK1775/MK8776 warrants further investigation as a potential treatment for acute myeloid leukemia.

Expression of Insulin Receptor Isoform A and Insulin-like Growth Factor-1 Receptor in Human Acute Myelogenous Leukemia: Effect of the Dual-Receptor Inhibitor BMS-536924 In vitro

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are receptor tyrosine kinases that participate in mitogenic and antiapoptotic signaling in normal and neoplastic epithelia. In the present study, immunoblotting and reverse transcription-PCR demonstrated expression of IGF1R and IR isoform A in acute myelogenous leukemia (AML) cell lines as well as in >80% of clinical AML isolates. Treatment with insulin enhanced signaling through the Akt and MEK1/2 pathways as well as survival of serum-starved AML cell lines. Conversely, treatment with BMS-536924, a dual IGF1R/IR kinase inhibitor that is undergoing preclinical testing, inhibited constitutive receptor phosphorylation as well as downstream signaling through MEK1/2 and Akt. These changes inhibited proliferation and, in some AML cell lines, induced apoptosis at submicromolar concentrations. Likewise, BMS-536924 inhibited leukemic colony formation in CD34+ clinical AML samples in vitro. Collectively, these results not only indicate that expression of IGF1R and IR isoform A is common in AML but also show that interruption of signaling from these receptors inhibits proliferation in clinical AML isolates. Accordingly, further investigation of IGF1R/IR axis as a potential therapeutic target in AML appears warranted.

Effects of Selective Checkpoint Kinase 1 Inhibition on Cytarabine Cytotoxicity in Acute Myelogenous Leukemia Cells In Vitro

Purpose: Previous studies have shown that the replication checkpoint, which involves the kinases ataxia telangiectasia mutated and Rad3 related (ATR) and Chk1, contributes to cytarabine resistance in cell lines. In the present study, we examined whether this checkpoint is activated in clinical acute myelogenous leukemia (AML) during cytarabine infusion in vivo and then assessed the impact of combining cytarabine with the recently described Chk1 inhibitor SCH 900776 in vitro. Experimental design: AML marrow aspirates harvested before and during cytarabine infusion were examined by immunoblotting. Human AML lines treated with cytarabine in the absence or presence of SCH 900776 were assayed for checkpoint activation by immunoblotting, nucleotide incorporation into DNA, and flow cytometry. Long-term effects in AML lines, clinical AML isolates, and normal myeloid progenitors were assayed using clonogenic assays. Results: Immunoblotting revealed increased Chk1 phosphorylation, a marker of checkpoint activation, in more than half of Chk1-containing AMLs after 48 hours of cytarabine infusion. In human AML lines, SCH 900776 not only disrupted cytarabine-induced Chk1 activation and S-phase arrest but also markedly increased cytarabine-induced apoptosis. Clonogenic assays demonstrated that SCH 900776 enhanced the antiproliferative effects of cytarabine in AML cell lines and clinical AML samples at concentrations that had negligible impact on normal myeloid progenitors. Conclusions: These results not only provide evidence for cytarabine-induced S-phase checkpoint activation in AML in the clinical setting, but also show that a selective Chk1 inhibitor can overcome the S-phase checkpoint and enhance the cytotoxicity of cytarabine. Accordingly, further investigation of the cytarabine/SCH 900776 combination in AML appears warranted. Clin Cancer Res; 18(19); 5364–73. ©2012 AACR.

Multi-institutional phase 2 study of the farnesyltransferase inhibitor tipifarnib (R115777) in patients with relapsed and refractory lymphomas

A phase 2 study of the oral farnesyltransferase inhibitor tipifarnib was conducted in 93 adult patients with relapsed or refractory lymphoma. Patients received tipifarnib 300 mg twice daily on days 1-21 of each 28-day cycle. The median number of prior therapies was 5 (range, 1-17). For the aggressive B-cell, indolent B-cell, and T-cell and Hodgkin lymphoma (HL/T) groups, the response rates were 17% (7/42), 7% (1/15), and 31% (11/36), respectively. Of the 19 responders, 7 were diffuse large B-cell non-Hodgkin lymphoma (NHL), 7 T-cell NHL, 1 follicular grade 2, and 4 HL. The median response duration for the 19 responders was 7.2 months (mean, 15.8 months; range, 1.8-62), and 5 patients in the HL/T group are still receiving treatment at 29-64+ months. The grade 3/4 toxicities observed were fatigue and reversible myelosuppression. Correlative studies suggest that Bim and Bcl-2 should be examined as potential predictors of response in future studies. These results indicate that tipifarnib has activity in lymphoma, particularly in heavily pretreated HL/T types, with little activity in follicular NHL. In view of its excellent toxicity profile and novel mechanism of action, further studies in combination with other agents appear warranted. This trial is registered at www.clinicaltrials.gov as #NCT00082888.

CXCR4 chemokine receptor signaling induces apoptosis in acute myeloid leukemia cells via regulation of the Bcl-2 family members Bcl-XL, Noxa, and Bak.

Background: The chemokine receptor CXCR4 plays a role in AML. Results: SDF-1, the ligand of CXCR4, induces apoptosis in AML cell lines and patient samples via modulation of Bcl-2 family members. Conclusion: SDF-1 induces apoptosis of AML cells via up-regulation of Bak and Noxa and down-regulation of Bcl-XL. Significance: SDF-1/CXCR4 signaling could induce AML cell apoptosis if bone marrow survival cues can be disrupted. The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted.

High Cell Surface Death Receptor Expression Determines Type I Versus Type II Signaling

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Phase I and pharmacological study of cytarabine and tanespimycin in relapsed and refractory acute leukemia.

Background In preclinical studies the heat shock protein 90 (Hsp90) inhibitor tanespimycin induced down-regulation of checkpoint kinase 1 (Chk1) and other client proteins as well as increased sensitivity of acute leukemia cells to cytarabine. We report here the results of a phase I and pharmacological study of the cytarabine + tanespimycin combination in adults with recurrent or refractory acute leukemia. Design and Methods Patients received cytarabine 400 mg/m2/day continuously for 5 days and tanespimycin infusions at escalating doses on days 3 and 6. Marrow mononuclear cells harvested before therapy, immediately prior to tanespimycin, and 24 hours later were examined by immunoblotting for Hsp70 and multiple Hsp90 clients. Results Twenty-six patients were treated at five dose levels. The maximum tolerated dose was cytarabine 400 mg/m2/day for 5 days along with tanespimycin 300 mg/m2 on days 3 and 6. Treatment-related adverse events included disseminated intravascular coagulation (grades 3 and 5), acute respiratory distress syndrome (grade 4), and myocardial infarction associated with prolonged exposure to tanespimycin and its active metabolite 17-aminogeldanamycin. Among 21 evaluable patients, there were two complete and four partial remissions. Elevations of Hsp70, a marker used to assess Hsp90 inhibition in other studies, were observed in more than 80% of samples harvested 24 hours after tanespimycin, but down-regulation of Chk1 and other Hsp90 client proteins was modest. Conclusions Because exposure to potentially effective concentrations occurs only for a brief time in vivo, at clinically tolerable doses tanespimycin has little effect on resistance-mediating client proteins in relapsed leukemia and exhibits limited activity in combination with cytarabine.