Dhruv Chachad

ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies

ONC201 triggers an apoptotic cellular stress response in both solid and blood tumors. Stressing cancer cells to death The anticancer drug ONC201 triggers cell death in various tumor types. A pair of papers (see also the Focus by Greer and Lipkowitz) shows that ONC201 activated cell stress pathways that depended on the activation of the transcription factor ATF4. Kline et al. showed that this stress response to ONC201 occurred in cells derived from various types of solid tumors, in which ATF4 activation led to an increase in the abundance of the proapoptotic protein TRAIL and its receptor DR5. Ishizawa et al. demonstrated that in acute myeloid leukemias and mantle cell lymphoma, ONC201 triggered apoptosis and inhibited mTORC1 signaling, a pathway that promotes cell growth and proliferation. The findings reveal more details about ONC201’s mechanism of action, potentially enabling patient stratification and future development to improve its efficacy. The clinical challenge posed by p53 abnormalities in hematological malignancies requires therapeutic strategies other than standard genotoxic chemotherapies. ONC201 is a first-in-class small molecule that activates p53-independent apoptosis, has a benign safety profile, and is in early clinical trials. We found that ONC201 caused p53-independent apoptosis and cell cycle arrest in cell lines and in mantle cell lymphoma (MCL) and acute myeloid leukemia (AML) samples from patients; these included samples from patients with genetic abnormalities associated with poor prognosis or cells that had developed resistance to the nongenotoxic agents ibrutinib and bortezomib. Moreover, ONC201 caused apoptosis in stem and progenitor AML cells and abrogated the engraftment of leukemic stem cells in mice while sparing normal bone marrow cells. ONC201 caused changes in gene expression similar to those caused by the unfolded protein response (UPR) and integrated stress responses (ISRs), which increase the translation of the transcription factor ATF4 through an increase in the phosphorylation of the translation initiation factor eIF2α. However, unlike the UPR and ISR, the increase in ATF4 abundance in ONC201-treated hematopoietic cells promoted apoptosis and did not depend on increased phosphorylation of eIF2α. ONC201 also inhibited mammalian target of rapamycin complex 1 (mTORC1) signaling, likely through ATF4-mediated induction of the mTORC1 inhibitor DDIT4. Overexpression of BCL-2 protected against ONC201-induced apoptosis, and the combination of ONC201 and the BCL-2 antagonist ABT-199 synergistically increased apoptosis. Thus, our results suggest that by inducing an atypical ISR and p53-independent apoptosis, ONC201 has clinical potential in hematological malignancies.

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs.

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.

Preclinical activity of the novel B-cell-specific Moloney murine leukemia virus integration site 1 inhibitor PTC-209 in acute myeloid leukemia: Implications for leukemia therapy

Curing patients with acute myeloid leukemia (AML) remains a therapeutic challenge. The polycomb complex protein B‐cell‐specific Moloney murine leukemia virus integration site 1 (BMI‐1) is required for the self‐renewal and maintenance of leukemia stem cells. We investigated the prognostic significance of BMI‐1 in AML and the effects of a novel small molecule selective inhibitor of BMI‐1, PTC‐209. BMI‐1 protein expression was determined in 511 newly diagnosed AML patients together with 207 other proteins using reverse‐phase protein array technology. Patients with unfavorable cytogenetics according to Southwest Oncology Group criteria had higher levels of BMI‐1 compared to those with favorable (P = 0.0006) or intermediate cytogenetics (P = 0.0061), and patients with higher levels of BMI‐1 had worse overall survival (55.3 weeks vs. 42.8 weeks, P = 0.046). Treatment with PTC‐209 reduced protein level of BMI‐1 and its downstream target mono‐ubiquitinated histone H2A and triggered several molecular events consistent with the induction of apoptosis, this is, loss of mitochondrial membrane potential, caspase‐3 cleavage, BAX activation, and phosphatidylserine externalization. PTC‐209 induced apoptosis in patient‐derived CD34+CD38low/− AML cells and, less prominently, in CD34− differentiated AML cells. BMI‐1 reduction by PTC‐209 directly correlated with apoptosis induction in CD34+ primary AML cells (r = 0.71, P = 0.022). However, basal BMI‐1 expression was not a determinant of AML sensitivity. BMI‐1 inhibition, which targets a primitive AML cell population, might offer a novel therapeutic strategy for AML.

FZR1 loss increases sensitivity to DNA damage and consequently promotes murine and human B-cell acute leukemia

FZR1 (fizzy-related protein homolog; also known as CDH1 [cell division cycle 20 related 1]) functions in the cell cycle as a specific activator of anaphase-promoting complex or cyclosome ubiquitin ligase, regulating late mitosis, G1 phase, and activation of the G2-M checkpoint. FZR1 has been implicated as both a tumor suppressor and oncoprotein, and its precise contribution to carcinogenesis remains unclear. Here, we examined the role of FZR1 in tumorigenesis and cancer therapy by analyzing tumor models and patient specimens. In an Fzr1 gene-trap mouse model of B-cell acute lymphoblastic leukemia (B-ALL), mice with Fzr1-deficient B-ALL survived longer than those with Fzr1-intact disease, and sensitivity of Fzr1-deficient B-ALL cells to DNA damage appeared increased. Consistently, conditional knockdown of FZR1 sensitized human B-ALL cell lines to DNA damage-induced cell death. Moreover, multivariate analyses of reverse-phase protein array of B-ALL specimens from newly diagnosed B-ALL patients determined that a low FZR1 protein expression level was an independent predictor of a longer remission duration. The clinical benefit of a low FZR1 expression level at diagnosis was no longer apparent in patients with relapsed B-ALL. Consistent with this result, secondary and tertiary mouse recipients of Fzr1-deficient B-ALL cells developed more progressive and radiation-resistant disease than those receiving Fzr1-intact B-ALL cells, indicating that prolonged inactivation of Fzr1 promotes the development of resistant clones. Our results suggest that reduction of FZR1 increases therapeutic sensitivity of B-ALL and that transient rather than tonic inhibition of FZR1 may be a therapeutic strategy.

Abstract B1: Gene expression and TP53 mutation analysis predict sensitivity of leukemia cells to MDM2 inhibition by DS-3032b

Background: MDM2 overexpression, by preventing p53 activation, contributes to the growth and development of a variety of solid tumors and hematologic malignancies; hence, MDM2 inhibition could be a promising novel therapeutic strategy. Several MDM2 inhibitors have shown promise in early clinical trials. While preclinical studies generally reveal a requirement of wild-type (wt) TP53 for activity, tumor response to MDM2 inhibitors varies widely in the clinic and may not be strictly linked to TP53 mutational status. Identification of predictive biomarkers is therefore needed to enrich for patients with high likelihood of response. We here propose two gene signature-based models to predict the sensitivity of AML cells to MDM2 inhibition using two different methods. Methods: Leukemia samples isolated from peripheral blood or bone marrow of patients with newly diagnosed or relapsed/refractory AML were treated using DS-3032b (Daiichi-Sankyo), a dispiropyrrolidine-based, highly potent MDM2 inhibitor currently undergoing clinical trials in solid and hematological malignancies. Forty-one primary AML samples were treated ex vivo for 48 hours with DS-3032b (0, 25, 50, 100, 250, 500, and 1000 nM), and live cell numbers were determined. To define drug sensitivity/resistance, area under the curve (AUC) values, based on%live cell number measured at each concentration, were calculated. Baseline whole-genome RNA expression profile (Affymetrix Human Genome U133 Plus 2.0 Array) and TP53 mutation status (next generation sequencing) were determined. In the first model, we validated a predictive 175-gene signature that was established in a wide range of cancer cells by Daiichi Sankyo. In the second model, we used the random forest method with cross validation to establish a new predictive gene signature. Results: Eight samples (20%) had TP53 mutations. 6/8 (75%) p53 mutant and 8/33 (24%) of p53 wt samples were resistant (p = 0.01). In the first model, 11 each p53 wt samples were selected as sensitive or resistant to DS-3032 based on AUC values, and the 175-gene signature was applied. The prediction accuracy was 72%. In the genotype mixed samples, 14 each sensitive and resistant samples were selected, and the prediction accuracy was 79%. In the second model, we focused on 33 p53 wt samples and trichotomize the samples in the same way as in the first model, and investigated the accuracy of gene expression-derived prediction model with (A) 1500 gene set with the highest variance in mRNA expression (unbiased approach), (B) 32 gene set derived from previous studies (referenced approach), (C) combined (A+B) gene set. The sensitivities to predict cases with high drug sensitivity were 72%, 73% and 82% in scenarios (A), (B) and (C), respectively. The analysis was then extended to all 41 samples and the sensitivity to predict cases with high drug sensitivity remained high (64%, 64% and 72%). The results indicate that an unbiased approach can create a prediction model as accurate as the referenced approach, and moreover, that the combining approach can provide the highest prediction of sensitivity to the MDM2 inhibitor. Conclusion: The two models reported here could provide a novel strategy to identify the optimal gene signatures for predicting the cases most sensitive to MDM2 inhibitors prior to therapy. These models will be tested in an ongoing AML phase 1 clinical study of DS-3032b. Citation Format: Jo Ishizawa, Kenji Nakamaru, Takahiko Seki, Koichi Tazaki, Kensuke Kojima, Dhruv Chachad, Archie Tse, Arvind Rao, Michael Andreeff. Gene expression and TP53 mutation analysis predict sensitivity of leukemia cells to MDM2 inhibition by DS-3032b. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B1.

Predictive Gene Signatures Determine Tumor Sensitivity to MDM2 Inhibition

Early clinical trials using murine double minute 2 (MDM2) inhibitors demonstrated proof-of-concept of p53-induced apoptosis by MDM2 inhibition in cancer cells; however, not all wild-type TP53 tumors are sensitive to MDM2 inhibition. Therefore, more potent inhibitors and biomarkers predictive of tumor sensitivity are needed. The novel MDM2 inhibitor DS-3032b is 10-fold more potent than the first-generation inhibitor nutlin-3a. TP53 mutations were predictive of resistance to DS-3032b, and allele frequencies of TP53 mutations were negatively correlated with sensitivity to DS-3032b. However, sensitivity to DS-3032b of TP53 wild-type tumors varied greatly. We thus used two methods to create predictive gene signatures. First, by comparing sensitivity to MDM2 inhibition with basal mRNA expression profiles in 240 cancer cell lines, a 175-gene signature was defined and validated in patient-derived tumor xenograft models and ex vivo human acute myeloid leukemia (AML) cells. Second, an AML-specific 1,532-gene signature was defined by performing random forest analysis with cross-validation using gene expression profiles of 41 primary AML samples. The combination of TP53 mutation status with the two gene signatures provided the best positive predictive values (81% and 82%, compared with 62% for TP53 mutation status alone). In addition, the top-ranked 50 genes selected from the AML-specific 1,532-gene signature conserved high predictive performance, suggesting that a more feasible size of gene signature can be generated through this method for clinical implementation. Our model is being tested in ongoing clinical trials of MDM2 inhibitors.Significance: This study demonstrates that gene expression profiling combined with TP53 mutational status predicts antitumor effects of MDM2 inhibitors in vitro and in vivoCancer Res; 78(10); 2721-31. ©2018 AACR.

Abstract 2938: BMI-1 inhibition by PTC-209 induces mitochondrial apoptosis in acute myeloid leukemia cells

Leukemia stem cells play important roles in leukemia initiation, progression, and relapse, and represent a critical target for therapeutic intervention. BMI-1 is essential for the self-renewal of normal hematopoietic and leukemia stem cells. High expression of BMI-1 has been associated with poor prognosis in AML. PTC-209 is a novel transcriptional inhibitor of BMI-1, which has been reported to exhibit antitumor activity against cancer-initiating cells in colorectal cancer. We investigated anti-leukemia effects of the BMI-1 inhibitor PTC-209 on AML cells. A total of 8 AML cell lines (MOLM-13, OCI-AML3, MV4-11, NB4, HL-60, U-937, MOLM-14, OCI-AML2) were treated with different concentrations of PTC-209 for 48 hours and the IC50 values (concentration at which cell growth is inhibited by 50% at 48 hours of exposure) and the ED50 values (effective concentration inducing 50% killing as measured by Annexin V positivity) were determined. The IC50 values were 0.38 ± 0.07 μM (mean ± SEM), indicating high anti-proliferation effect. The ED50 values ranged from 1.2 to 2.6 μM in 7 cell lines (1.97 ± 0.22 μM) and the remaining cell line (NB4) showed relative resistance to PTC-induced apoptosis (> 10 μM). PTC-209 exhibited dose- and time-dependent anti-proliferative and cytotoxic activities, by inducing G1-S transition delay and apoptosis. PTC-209 induced conformational change of BAX (i.e., BAX activation), loss of mitochondrial membrane potential (MMP), caspase-3 activation and DNA fragmentation in addition to phosphatidylserine (PS) externalization, indicating mitochondrial apoptosis. PTC-209 induced Annexin V in primary AML cells (23.4 ± 4.6% after 48-hour treatment with 1 μM PTC-209, n = 23) in a dose-dependent manner, which is more prominent in immature CD34+CD38- population (33.7 ± 6.0%; P = 0.0036). In accordance with its high anti-leukemia activity, PTC-209 strongly reduced cellular BMI-1 levels in CD34+CD38- AML cells. Higher reduction of BMI-1 expression was positively correlated with higher susceptibility to PTC-209 in CD34+CD38- AML cells (r = 0.88; P = 0.047). The apoptotic activity was observed independent of p53 or FLT3 mutational status of the leukemia cells. Our data suggest that BMI-1 inhibition by small molecule inhibitors may be developed into a novel therapeutic strategy for AML. (Drs M. Andreeff and S. Kimura are co-senior authors with equal contribution to the work) Citation Format: Kensuke Kojima, Yuki Nishida, Aya Maeda, Dhruv Chachad, Hiroaki Kitamura, Jo Ishizawa, Michael Andreeff, Shinya Kimura. BMI-1 inhibition by PTC-209 induces mitochondrial apoptosis in acute myeloid leukemia 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 2938. doi:10.1158/1538-7445.AM2015-2938