Amy Saur Conway

Activity of a selective inhibitor of nuclear export, selinexor (KPT-330), against AML-initiating cells engrafted into immunosuppressed NSG mice.

Currently available combination chemotherapy for acute myeloid leukemia (AML) often fails to result in long-term remissions, emphasizing the need for novel therapeutic strategies. We reasoned that targeted inhibition of a prominent nuclear exporter, XPO1/CRM1, could eradicate self-renewing leukemia-initiating cells (LICs) whose survival depends on timely XPO1-mediated transport of specific protein and RNA cargoes. Using an immunosuppressed mouse model bearing primary patient-derived AML cells, we demonstrate that selinexor (KPT-330), an oral antagonist of XPO1 that is currently in clinical trials, has strong activity against primary AML cells while sparing normal stem and progenitor cells. Importantly, limiting dilution transplantation assays showed that this cytotoxic activity is not limited to the rapidly proliferating bulk population of leukemic cells but extends to the LICs, whose inherent drug resistance and unrestricted self-renewal capacity has been implicated in the difficulty of curing AML patients with conventional chemotherapy alone.

Synergistic Drug Combinations with a CDK4/6 Inhibitor in T-cell Acute Lymphoblastic Leukemia

Purpose: Although significant progress has been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL), many patients will require additional therapy for relapsed/refractory disease. Cyclin D3 (CCND3) and CDK6 are highly expressed in T-ALL and have been effectively targeted in mutant NOTCH1-driven mouse models of this disease with a CDK4/6 small-molecule inhibitor. Combination therapy, however, will be needed for the successful treatment of human disease. Experimental Design: We performed preclinical drug testing using a panel of T-ALL cell lines first with LEE011, a CDK4/6 inhibitor, and next with the combination of LEE011 with a panel of drugs relevant to T-ALL treatment. We then tested the combination of LEE011 with dexamethasone or everolimus in three orthotopic mouse models and measured on-target drug activity. Results: We first determined that both NOTCH1-mutant and wild-type T-ALL are highly sensitive to pharmacologic inhibition of CDK4/6 when wild-type RB is expressed. Next, we determined that CDK4/6 inhibitors are antagonistic when used either concurrently or in sequence with many of the drugs used to treat relapsed T-ALL (methotrexate, mercaptopurine, asparaginase, and doxorubicin) but are synergistic with glucocorticoids, an mTOR inhibitor, and gamma secretase inhibitor. The combinations of LEE011 with the glucocorticoid dexamethasone or the mTOR inhibitor everolimus were tested in vivo and prolonged survival in three orthotopic mouse models of T-ALL. On-target activity was measured in peripheral blood and tissue of treated mice. Conclusions: We conclude that LEE011 is active in T-ALL and that combination therapy with corticosteroids and/or mTOR inhibitors warrants further investigation. Clin Cancer Res; 23(4); 1012–24. ©2016 AACR. See related commentary by Carroll et al., p. 873

KPT-8602, a second-generation inhibitor of XPO1-mediated nuclear export, is well tolerated and highly active against AML blasts and leukemia-initiating cells.

Acute myeloid leukemia (AML) is a clonal hematologic malignant disease of developing myeloid cells that have acquired aberrant survival, uncontrolled proliferation and a block in normal hematopoietic cell differentiation. Standard chemotherapy often induces remissions in AML patients, but the disease frequently relapses due to incomplete targeting of leukemia-initiating cells (LICs), emphasizing the need for novel effective treatments. Exportin 1 (XPO1)-mediated nuclear export, which is inhibited by the drug selinexor, is an attractive new therapeutic target in AML. Selinexor has shown impressive activity in Phase I/II clinical trials for AML. Here we report the anti-leukemic efficacy and tolerability of KPT-8602, a second-generation XPO1 inhibitor. KPT-8602 demonstrates substantially reduced brain penetration compared to selinexor, with resultant attenuation of the central nervous system mediated side effects of anorexia and weight loss. Due to its improved tolerability profile, KPT-8602 can be given daily compared to the two or three times weekly regimen of selinexor, and exhibits greater anti-leukemic efficacy against both leukemic blasts and LICs in AML patient-derived xenograft models. Importantly, normal hematopoietic stem and progenitor cell (HSPC) frequency is not significantly reduced by KPT-8602, providing a therapeutic window for elimination of relapse-driving LICs while sparing normal HSPCs. These findings strongly endorse clinical testing of KPT-8602 in patients with relapsed and refractory AML.

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

Expression of the MECOM (also known as EVI1) proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A screen using pooled short hairpin RNAs (shRNAs) identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as necessary for survival of EVI1-expressing cells in subjects with EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine–creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted the cell cycle arrest and apoptosis of human EVI1-positive cell lines, and prolonged survival in both orthotopic xenograft models and mouse models of primary AML. CKMT1 inhibition altered mitochondrial respiration and ATP production, an effect that was abrogated by phosphocreatine-mediated reactivation of the arginine–creatine pathway. Targeting CKMT1 is thus a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens.

CRISPR-Cas9 screen reveals a MYCN -amplified neuroblastoma dependency on EZH2

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma.

Leukemia-specific delivery of mutant NOTCH1 targeted therapy

On-target drug delivery remains a challenge in cancer precision medicine; it is difficult to deliver a targeted therapy to cancer cells without incurring toxicity to normal tissues. The SERCA (sarco-endoplasmic reticulum Ca2+ ATPase) inhibitor thapsigargin inhibits mutant NOTCH1 receptors compared with wild type in T cell acute lymphoblastic leukemia (T-ALL), but its administration is predicted to be toxic in humans. Leveraging the addiction of ALL to folic acid, we conjugated folate to an alcohol derivative of thapsigargin via a cleavable ester linkage. JQ-FT is recognized by folate receptors on the plasma membrane and delivered into leukemia cells as a potent antileukemic agent. In mechanistic and translational models of T-ALL, we demonstrate NOTCH1 inhibition in vitro and in vivo. These proof-of-concept studies support the further optimization of this first-in-class NOTCH1 inhibitor with dual selectivity: leukemia over normal cells and NOTCH1 mutants over wild-type receptors. Furthermore, tumor-specific disruption of Notch signaling may overcome legitimate concerns associated with the tumor suppressor function of nontargeted Notch pathway inhibitors.

Genome-scale CRISPR-Cas9 screen identifies druggable dependencies in TP53 wild-type Ewing sarcoma

Ewing sarcoma is a pediatric cancer driven by EWS-ETS transcription factor fusion oncoproteins in an otherwise stable genomic background. The majority of tumors express wild-type TP53, and thus, therapies targeting the p53 pathway would benefit most patients. To discover targets specific for TP53 wild-type Ewing sarcoma, we used a genome-scale CRISPR-Cas9 screening approach and identified and validated MDM2, MDM4, USP7, and PPM1D as druggable dependencies. The stapled peptide inhibitor of MDM2 and MDM4, ATSP-7041, showed anti-tumor efficacy in vitro and in multiple mouse models. The USP7 inhibitor, P5091, and the Wip1/PPM1D inhibitor, GSK2830371, decreased the viability of Ewing sarcoma cells. The combination of ATSP-7041 with P5091, GSK2830371, and chemotherapeutic agents showed synergistic action on the p53 pathway. The effects of the inhibitors, including the specific USP7 inhibitor XL-188, were rescued by concurrent TP53 knockout, highlighting the essentiality of intact p53 for the observed cytotoxic activities.

Abstract 1118: Synthetic lethality of CDK12 inhibition in tumors with EWS/FLI rearrangements

THZ1 is a potent, covalent inhibitor of the transcriptional CDKs, CDK7/12/13. Chemical genomic profiling of THZ1 across >1,000 diverse cancer cell lines revealed that EWS/FLI- rearranged Ewing sarcoma cells were remarkably sensitive to this molecule. We demonstrated that THZ1 inhibits the phosphorylation of the C-terminal domain of RNA Polymerase II, decreased colony formation capacity, and induced apoptosis in a dose-dependent manner in Ewing sarcoma cell lines. Using selective CDK7 and CDK12/13 inhibitors, we revealed that the primary target of THZ1 in Ewing sarcoma is CDK12/13. Genetic suppression of CDK12, but not CDK13, induced strong anti-viability effects, confirming CDK12 as the primary target. Treatment of Ewing sarcoma cell lines with THZ531, a novel CDK12/13 selective inhibitor, preferentially repressed genes involved in DNA damage repair. Additionally, suppression of EWS/FLI rendered Ewing sarcoma cells resistant to THZ531 and partially rescued the anti-viability effects of CDK12 knockdown. These results suggest that EWS/FLI imparts vulnerability to DNA damage repair inhibition and implicate a synthetic lethal relationship between the tumor-specific expression of EWS/FLI and CDK12 inhibition. Furthermore, we demonstrated that CDK12 and PARP inhibitors are highly synergistic in vitro, inducing widespread yH2AX foci formation. Interestingly, THZ531 impairs the ability of the PARP inhibitor, olaparib, to induce RAD51 foci formation, suggesting that THZ531 specifically causes a defect in homologous recombination repair. Moreover, we observed striking synergy of THZ1 and olaparib in two mouse models of Ewing sarcoma with limited toxicity observed. These findings have important translational significance as clinical trials with PARP inhibitors as single agents in Ewing sarcoma failed to demonstrate efficacy, highlighting the need to identify combination therapies that will enhance the activity of PARP inhibition. We anticipate that CDK12 and PARP inhibitor combinations will be of therapeutic interest in other ETS-rearranged tumors, as well as tumors with defects in DNA repair. Citation Format: Amanda L. Balboni, Bjorn Stolte, Amy Saur Conway, Gabriela Alexe, Emily Jue Wang, Nicholas Kwiatkowski, Tinghu Zhang, Brian J. Abraham, Peter Kalev, Dipanjan Chowdhury, Cyril H. Benes, Richard A. Young, Nathanael S. Gray, Kimberly Stegmaier. Synthetic lethality of CDK12 inhibition in tumors with EWS/FLI rearrangements [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1118. doi:10.1158/1538-7445.AM2017-1118

Abstract 4445: Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells

Current treatments for acute myeloid leukemia (AML) often fail to induce long-term remissions and are toxic to normal tissues, prompting the need to develop new targeted therapies. The frequent disease relapse that is observed in patients with AML is thought to occur because of the inability of the existing drugs to target the self-renewing leukemia-initiating cells (LICs). An attractive new strategy for AML therapy is inhibition of the nuclear export protein exporter 1 (XPO1), or CRM1. XPO1 regulates export of proteins that contain leucine-rich nuclear export signals (NES), including protein adaptors that mediate transport of RNA. XPO1 cargo encompass tumor suppressor proteins, cell cycle regulators, and apoptotic proteins. Recently, small molecule inhibitors of nuclear export (SINE) that inhibit the export function of XPO1 by targeting Cys528 in its NES-binding groove, were developed using an in silico molecular modeling. Selinexor (KPT-330), the orally bioavailable SINE compound, is in Phase 1 and 2 studies in adult patients with AML (NCT01607892 and NCT02088541) and in a Phase 1 study for relapsed childhood ALL and AML initiated in March 2014 (NCT02091245). To define the anti-leukemic activity of selinexor against primary AML blasts and LICs in a clinically relevant setting, we established mouse models of primary human leukemia, or patient-derived xenografts (PDX), in which leukemic blasts from AML patients were transplanted into immunodeficient NOD-SCID-IL2Rcγnull (NSG) mice. Mice engrafted with leukemic blasts were treated with either vehicle or selinexor. Selinexor was highly active against blast cells from two of the three patients with poor-prognosis disease (cytogenetically normal AML with FLT3-ITD (AML-CN) and complex karyotype AML (AML-CK1 and AML-CK2)), as evidenced by a reduction in leukemic engraftment in primary mice after 4 weeks of treatment. Secondary transplantation assays indicated that selinexor greatly reduced the frequency of LICs in PDX models derived from all three patients (6- to 430- fold reduction compared to controls), indicating that this agent not only targets the bulk leukemic cells, but also eliminates LICs. These findings show that selinexor has potent activity against LICs, even when it has only moderate activity against the bulk AML cell population. Furthermore, preliminary results of combination studies of selinexor with Ara-C, a standard chemotherapeutic agent, demonstrate synergistic effect of the two drugs against LICs in a PDX model of AML-CN. Importantly, 4 weeks of selinexor treatment demonstrated minimal toxicity in mice engrafted with normal human CD34+ hematopoietic cells. These findings demonstrate that inhibition of nuclear export with selinexor overcomes an important obstacle to cure of AML, which is to destroy the very critical LIC compartment while sparing normal hematopoietic cells. Citation Format: Julia Etchin, Bonnie Thi Le, Alla Berezovskaya, Amy S. Conway, Weihsu C. Chen, Alex Kentsis, Marc R. Mansour, Richard M. Stone, Ilene A. Galinsky, Daniel J. DeAngelo, Dilara McCauley, Michael Kauffman, Sharon Shacham, Jean CY Wang, Andrew L. Kung, Thomas Look. Selective inhibitor of nuclear exporter CRM1/XPO1, Selinexor (KPT-330), exhibits remarkable activity against AML leukemia-initiating cells while sparing normal hematopoietic cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4445. doi:10.1158/1538-7445.AM2015-4445