Paula A. Schneider

MCL-1 as a Buffer for Proapoptotic BCL-2 Family Members during TRAIL-induced Apoptosis A MECHANISTIC BASIS FOR SORAFENIB (BAY 43-9006)-INDUCED TRAIL SENSITIZATION

Previous studies have suggested that Mcl-1, an antiapoptotic Bcl-2 homolog that does not exhibit appreciable affinity for the caspase 8-generated C-terminal Bid fragment (tBid), diminishes sensitivity to tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL). This study was performed to determine the mechanism by which Mcl-1 confers TRAIL resistance and to evaluate methods for overcoming this resistance. Affinity purification/immunoblotting assays using K562 human leukemia cells, which contain Mcl-1 and Bcl-xL as the predominant antiapoptotic Bcl-2 homologs, demonstrated that TRAIL treatment resulted in binding of tBid to Bcl-xL but not Mcl-1. In contrast, TRAIL caused increased binding between Mcl-1 and Bak that was diminished by treatment with the caspase 8 inhibitor N-(Nα-acetylisoleucylglutamylthreonyl) aspartic acid (O-methyl ester)-fluoromethyl ketone (IETD(OMe)-fmk) or the c-Jun N-terminal kinase inhibitor SP600125. In addition, TRAIL caused increased binding of Bim and Puma to Mcl-1 that was inhibited by IETD(OMe)-fmk but not SP600125. Further experiments demonstrated that down-regulation of Mcl-1 by short hairpin RNA or the kinase inhibitor sorafenib increased TRAIL-induced Bak activation and death ligand-induced apoptosis in a wide variety of neoplastic cell lines as well as clinical acute myelogenous leukemia specimens. Collectively, these observations not only suggest a model in which Mcl-1 confers TRAIL resistance by serving as a buffer for Bak, Bim, and Puma, but also identify sorafenib as a potential modulator of TRAIL sensitivity.

Transient binding of an activator BH3 domain to the Bak BH3-binding groove initiates Bak oligomerization

Interaction between the hydrophobic Bak BH3-binding groove and the BH3 domain of activator proteins is a key step in initiating Bak oligomerization and activation.

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.

Enhanced Killing of Cancer Cells by Poly(ADP-ribose) Polymerase Inhibitors and Topoisomerase I Inhibitors Reflects Poisoning of Both Enzymes

Background: PARP inhibitors and topoisomerase I poisons (Top1p) synergize by an unknown mechanism. Results: Although Parp1 deletion fails to increase Top1p sensitivity, transfection with catalytically inactive PARP1 or its isolated DNA binding domain does sensitize. Conclusion: PARP inhibitors poison PARP1 to diminish repair of topoisomerase I-triggered DNA damage. Significance: These results predict that tumors with elevated PARP1 will be particularly sensitive to Top1p/PARP inhibitor combinations. Poly(ADP-ribose) polymerase-1 (PARP1) plays critical roles in the regulation of DNA repair. Accordingly, small molecule inhibitors of PARP are being developed as agents that could modulate the activity of genotoxic chemotherapy, such as topoisomerase I poisons. In this study we evaluated the ability of the PARP inhibitor veliparib to enhance the cytotoxicity of the topoisomerase I poisons topotecan and camptothecin (CPT). Veliparib increased the cell cycle and cytotoxic effects of topotecan in multiple cell line models. Importantly, this sensitization occurred at veliparib concentrations far below those required to substantially inhibit poly(ADP-ribose) polymer synthesis and at least an order of magnitude lower than those involved in selective killing of homologous recombination-deficient cells. Further studies demonstrated that veliparib enhanced the effects of CPT in wild-type mouse embryonic fibroblasts (MEFs) but not Parp1−/− MEFs, confirming that PARP1 is the critical target for this sensitization. Importantly, parental and Parp1−/− MEFs had indistinguishable CPT sensitivities, ruling out models in which PARP1 catalytic activity plays a role in protecting cells from topoisomerase I poisons. To the contrary, cells were sensitized to CPT in a veliparib-independent manner upon transfection with PARP1 E988K, which lacks catalytic activity, or the isolated PARP1 DNA binding domain. These results are consistent with a model in which small molecule inhibitors convert PARP1 into a protein that potentiates the effects of topoisomerase I poisons by binding to damaged DNA and preventing its normal repair.

Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide

The farnesyltransferase inhibitor tipifarnib exhibits modest activity against acute myelogenous leukemia. To build on these results, we examined the effect of combining tipifarnib with other agents. Tipifarnib inhibited signaling downstream of the farnesylated small G protein Rheb and synergistically enhanced etoposide-induced antiproliferative effects in lymphohematopoietic cell lines and acute myelogenous leukemia isolates. We subsequently conducted a phase 1 trial of tipifarnib plus etoposide in adults over 70 years of age who were not candidates for conventional therapy. A total of 84 patients (median age, 77 years) received 224 cycles of oral tipifarnib (300-600 mg twice daily for 14 or 21 days) plus oral etoposide (100-200 mg daily on days 1-3 and 8-10). Dose-limiting toxicities occurred with 21-day tipifarnib. Complete remissions were achieved in 16 of 54 (30%) receiving 14-day tipifarnib versus 5 of 30 (17%) receiving 21-day tipifarnib. Complete remissions occurred in 50% of two 14-day tipifarnib cohorts: 3A (tipifarnib 600, etoposide 100) and 8A (tipifarnib 400, etoposide 200). In vivo, tipifarnib plus etoposide decreased ribosomal S6 protein phosphorylation and increased histone H2AX phosphorylation and apoptosis. Tipifarnib plus etoposide is a promising orally bioavailable regimen that warrants further evaluation in elderly adults who are not candidates for conventional induction chemotherapy. These clinical studies are registered at www.clinicaltrials.gov as #NCT00112853.

BCL2 mutations are associated with increased risk of transformation and shortened survival in follicular lymphoma

Follicular lymphoma (FL), an indolent neoplasm caused by a t(14;18) chromosomal translocation that juxtaposes the BCL2 gene and immunoglobulin locus, has a variable clinical course and frequently undergoes transformation to an aggressive lymphoma. Although BCL2 mutations have been previously described, their relationship to FL progression remains unclear. In this study, we evaluated the frequency and nature of BCL2 mutations in 2 independent cohorts of grade 1 and 2 FLs, along with the correlation between BCL2 mutations, transformation risk, and survival. The prevalence of BCL2 coding sequence mutations was 12% in FL at diagnosis and 53% at transformation (P < .0001). The presence of these BCL2 mutations at diagnosis correlated with an increased risk of transformation (hazard ratio 3.6; 95% CI, 2.0-6.2; P < .0001) and increased risk of death due to lymphoma (median survival of 9.5 years with BCL2 mutations vs 20.4 years without; P = .012). In a multivariate analysis, BCL2 mutations and high FL international prognostic index were independent risk factors for transformation and death due to lymphoma. Some mutant Bcl-2 proteins exhibited enhanced antiapoptotic capacity in vitro. Accordingly, BCL2 mutations can affect antiapoptotic Bcl-2 function, are associated with increased activation-induced cytidine deaminase expression, and correlate with increased risk of transformation and death due to lymphoma.

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.

Context-dependent Bcl-2/Bak interactions regulate lymphoid cell apoptosis

The release of cytochrome c from mitochondria, which leads to activation of the intrinsic apoptotic pathway, is regulated by interactions of Bax and Bak with antiapoptotic Bcl-2 family members. The factors that regulate these interactions are, at the present time, incompletely understood. Recent studies showing preferences in binding between synthetic Bcl-2 homology domain 3 and antiapoptotic Bcl-2 family members in vitro have suggested that the antiapoptotic proteins Mcl-1 and Bcl-xL, but not Bcl-2, restrain proapoptotic Bak from inducing mitochondrial membrane permeabilization and apoptosis. Here we show that Bak protein has a much higher affinity than the 26-amino acid Bak Bcl-2 homology domain 3 for Bcl-2, that some naturally occurring Bcl-2 allelic variants have an affinity for full-length Bak that is only 3-fold lower than that of Mcl-1, and that endogenous levels of these Bcl-2 variants (which are as much as 40-fold more abundant than Mcl-1) restrain part of the Bak in intact lymphoid cells. In addition, we demonstrate that Bcl-2 variants can, depending on their affinity for Bak, substitute for Mcl-1 in protecting cells. Thus, the ability of Bcl-2 to protect cells from activated Bak depends on two important contextual variables, the identity of the Bcl-2 present and the amount expressed.

Bak Conformational Changes Induced by Ligand Binding: Insight into BH3 Domain Binding and Bak Homo-Oligomerization

Recently we reported that the BH3-only proteins Bim and Noxa bind tightly but transiently to the BH3-binding groove of Bak to initiate Bak homo-oligomerization. However, it is unclear how such tight binding can induce Bak homo-oligomerization. Here we report the ligand-induced Bak conformational changes observed in 3D models of Noxa·Bak and Bim·Bak refined by molecular dynamics simulations. In particular, upon binding to the BH3-binding groove, Bim and Noxa induce a large conformational change of the loop between helices 1 and 2 and in turn partially expose a remote groove between helices 1 and 6 in Bak. These observations, coupled with the reported experimental data, suggest formation of a pore-forming Bak octamer, in which the BH3-binding groove is at the interface on one side of each monomer and the groove between helices 1 and 6 is at the interface on the opposite side, initiated by ligand binding to the BH3-binding groove.