Leah Hogdal

Selective BCL-2 Inhibition by ABT-199 Causes On-Target Cell Death in Acute Myeloid Leukemia

B-cell leukemia/lymphoma 2 (BCL-2) prevents commitment to programmed cell death at the mitochondrion. It remains a challenge to identify those tumors that are best treated by inhibition of BCL-2. Here, we demonstrate that acute myeloid leukemia (AML) cell lines, primary patient samples, and murine primary xenografts are very sensitive to treatment with the selective BCL-2 antagonist ABT-199. In primary patient cells, the median IC50 was approximately 10 nmol/L, and cell death occurred within 2 hours. Our ex vivo sensitivity results compare favorably with those observed for chronic lymphocytic leukemia, a disease for which ABT-199 has demonstrated consistent activity in clinical trials. Moreover, mitochondrial studies using BH3 profiling demonstrate activity at the mitochondrion that correlates well with cytotoxicity, supporting an on-target mitochondrial mechanism of action. Our protein and BH3 profiling studies provide promising tools that can be tested as predictive biomarkers in any clinical trial of ABT-199 in AML.

Efficacy and Biological Correlates of Response in a Phase II Study of Venetoclax Monotherapy in Patients with Acute Myelogenous Leukemia

We present a phase II, single-arm study evaluating 800 mg daily venetoclax, a highly selective, oral small-molecule B-cell leukemia/lymphoma-2 (BCL2) inhibitor in patients with high-risk relapsed/refractory acute myelogenous leukemia (AML) or unfit for intensive chemotherapy. Responses were evaluated following revised International Working Group (IWG) criteria. The overall response rate was 19%; an additional 19% of patients demonstrated antileukemic activity not meeting IWG criteria (partial bone marrow response and incomplete hematologic recovery). Twelve (38%) patients had isocitrate dehydrogenase 1/2 mutations, of whom 4 (33%) achieved complete response or complete response with incomplete blood count recovery. Six (19%) patients had BCL2-sensitive protein index at screening, which correlated with time on study. BH3 profiling was consistent with on-target BCL2 inhibition and identified potential resistance mechanisms. Common adverse events included nausea, diarrhea and vomiting (all grades), and febrile neutropenia and hypokalemia (grade 3/4). Venetoclax demonstrated activity and acceptable tolerability in patients with AML and adverse features. SIGNIFICANCE Venetoclax monotherapy demonstrated clinical activity in patients with AML (relapsed/refractory or unfit for intensive chemotherapy) with a tolerable safety profile in this phase II study. Predictive markers of response consistent with BCL2 dependence were identified. Clinical and preclinical findings provide a compelling rationale to evaluate venetoclax combined with other agents in AML. Cancer Discov; 6(10); 1106-17. ©2016 AACRSee related commentary by Pullarkat and Newman, p. 1082This article is highlighted in the In This Issue feature, p. 1069.

Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells.

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.

MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199

Summary Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.

Blastic Plasmacytoid Dendritic Cell Neoplasm Is Dependent on BCL2 and Sensitive to Venetoclax

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy with dismal outcomes for which no standard therapy exists. We found that primary BPDCN cells were dependent on the antiapoptotic protein BCL2 and were uniformly sensitive to the BCL2 inhibitor venetoclax, as measured by direct cytotoxicity, apoptosis assays, and dynamic BH3 profiling. Animals bearing BPDCN patient-derived xenografts had disease responses and improved survival after venetoclax treatment in vivo Finally, we report on 2 patients with relapsed/refractory BPDCN who received venetoclax off-label and experienced significant disease responses. We propose that venetoclax or other BCL2 inhibitors undergo expedited clinical evaluation in BPDCN, alone or in combination with other therapies. In addition, these data illustrate an example of precision medicine to predict treatment response using ex vivo functional assessment of primary tumor tissue, without requiring a genetic biomarker. SIGNIFICANCE Therapy for BPDCN is inadequate, and survival in patients with the disease is poor. We used primary tumor cell functional profiling to predict BCL2 antagonist sensitivity as a common feature of BPDCN, and demonstrated in vivo clinical activity of venetoclax in patient-derived xenografts and in 2 patients with relapsed chemotherapy-refractory disease. Cancer Discov; 7(2); 156-64. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

Discovery and biological characterization of potent myeloid cell leukemia‐1 inhibitors

Myeloid cell leukemia 1 (Mcl‐1) is an antiapoptotic member of the Bcl‐2 family of proteins that when overexpressed is associated with high tumor grade, poor survival, and resistance to chemotherapy. Mcl‐1 is amplified in many human cancers, and knockdown of Mcl‐1 using RNAi can lead to apoptosis. Thus, Mcl‐1 is a promising cancer target. Here, we describe the discovery of picomolar Mcl‐1 inhibitors that cause caspase activation, mitochondrial depolarization, and selective growth inhibition. These compounds represent valuable tools to study the role of Mcl‐1 in cancer and serve as useful starting points for the discovery of clinically useful Mcl‐1 inhibitors.

Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways.

Targeting leukemogenic mutations identified by functional single-cell genomics eliminated human AML in vivo. The right treatments for the right mutations A variety of mutations have been observed in cancer cells from patients with acute myeloid leukemia, but it can be difficult to know which of these mutations contribute to tumorigenesis and should therefore be targeted. To address this issue, Saito et al. isolated subpopulations of leukemic cells with specific mutations and monitored their leukemogenic capacity in immunosuppressed mice. By combining this approach with genomic analysis, the authors were able to identify mutations that drive the evolution of leukemia and figure out effective approaches to target them. Numerous variant alleles are associated with human acute myeloid leukemia (AML). However, the same variants are also found in individuals with no hematological disease, making their functional relevance obscure. Through NOD.Cg-PrkdcscidIl2rgtmlWjl/Sz (NSG) xenotransplantation, we functionally identified preleukemic and leukemic stem cell populations present in FMS-like tyrosine kinase 3 internal tandem duplication–positive (FLT3-ITD)+ AML patient samples. By single-cell DNA sequencing, we identified clonal structures and linked mutations with in vivo fates, distinguishing mutations permissive of nonmalignant multilineage hematopoiesis from leukemogenic mutations. Although multiple somatic mutations coexisted at the single-cell level, inhibition of the mutation strongly associated with preleukemic to leukemic stem cell transition eliminated AML in vivo. Moreover, concurrent inhibition of BCL-2 (B cell lymphoma 2) uncovered a critical dependence of resistant AML cells on antiapoptotic pathways. Co-inhibition of pathways critical for oncogenesis and survival may be an effective strategy that overcomes genetic diversity in human malignancies. This approach incorporating single-cell genomics with the NSG patient-derived xenograft model may serve as a broadly applicable resource for precision target identification and drug discovery.

Abstract 2834: BH3 profiling predicts clinical response in a phase II clinical trial of ABT-199 (GDC-0199) in acute myeloid leukemia

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: ABT-199 is a potent and selective small molecule inhibitor of the anti-apoptotic protein BCL-2, currently in a Phase II clinical trial for relapsed/refractory acute myeloid leukemia (AML). Results from the trial indicate that AML patients have a heterogeneous response to ABT-199 used as a single agent. Therefore, it would be beneficial to prospectively identify patients who will be most sensitive to ABT-199 treatment. We have developed BH3 profiling, which is a rapid, functional assay that measures dependence on any of the anti-apoptotic BCL-2 family proteins for an individual sample. We have previously shown in many cancer cell lines and primary samples that BH3 profiling predicts in vitro sensitivity to ABT-199. Here, we test for correlation between clinical response to ABT-199 and BH3 profiling. Methods: Twelve pre-treatment AML bone marrow or peripheral blood samples were BH3 profiled using a panel of peptides which included BAD (measures BCL-2, BCL-XL, BCL-W dependence), MS1 (MCL-1 dependence), HRK (BCL-XL dependence) and mitochondrial ABT-199 (BCL-2 dependence). In addition, three samples obtained after 4 wks on study were also BH3 profiled to assess the in vivo mechanism of action of ABT-199. Results: BH3 profiling predicted clinical response in several ways. BCL-2 dependence predicted reduction of blast count at 4 wks, with an AUC of the ROC of 0.86. Myeloblast dependence on the anti-apoptotic protein MCL-1 or BCL-XL alone predicted low blast count reduction at 4 wks, with an AUC of the ROC of 0.80 and 0.97 respectively. Since dependence on MCL-1 or BCL-XL both predict poor clinical performance, we asked whether we could combine the information to make a superior predictor by arithmetically adding response to HRK to that to MS1. We found that this metric was a perfect binary predictor of blast reduction at 4 wks (AUC = 1.0). Additionally, we asked whether ABT-199 was working on-target, at the mitochondria in vivo. We BH3 profiled bone marrow aspirates collected while patients were on treatment and analyzed the response to the MS1, HRK and BIM peptides. We found that the AML blasts remaining following 4 wks of treatment have an increased response to the HRK, MS1 and BIM peptides compared to pre-treatment samples. This suggests that ABT-199 is working on target as it displaces pro-apoptotic proteins from BCL-2 to BCL-XL and MCL-1 and diminishes the overall anti-apoptotic reserve at the mitochondrion. Conclusions: The results of this study indicate that BH3 profiling can be used to exclude AML patients destined to respond poorly and enrich for those patients destined to respond well to single agent ABT-199. Since we have previously found that the level of mitochondrial apoptotic priming predicts clinical response to conventional chemotherapy, we speculate that ABT-199 could facilitate myeloblast killing via conventional chemotherapy in vivo. Citation Format: Leah Hogdal, Brenda Chyla, Evelyn McKeegan, Joel Leverson, Jalaja Potluri, Nancy Falotico, Justin Ricker, Rod Humerickhouse, Mack Mabry, Glenna Foight, Amy Keating, Ilene Galinsky, Richard Stone, Daniel DeAngelo, Marina Konopleva, Anthony Letai. BH3 profiling predicts clinical response in a phase II clinical trial of ABT-199 (GDC-0199) in acute myeloid leukemia. [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 2834. doi:10.1158/1538-7445.AM2015-2834

Abstract 3727: Small molecule Mcl-1 inhibitors induce apoptosis and death in multiple cancer subtypes in vitro

Mcl-1 is a member of the Bcl-2 family of proteins that play a major role in conferring resistance to apoptosis in cancer cells. Inhibiting Mcl-1 using peptides or RNAi has been shown to induce apoptosis in a broad array of cancer cell lines in numerous studies, making Mcl-1 a compelling target for anticancer therapy. We have discovered potent and selective small molecule Mcl-1 inhibitors that bind to the BH3 binding site of Mcl-1 with sub nanomolar affinities. These agents rapidly induced apoptosis in the Mcl-1 dependent NCI-H929 myeloma cell line, as demonstrated by mitochondrial membrane depolarization, caspase activation, and decreased viability. We measured the anti-proliferative activity of our compounds in cell lines from several cancer subtypes and found a broad spectrum of sensitivity to Mcl-1 inhibition. In cell lines that were resistant to Mcl-1 antagonists, combination with the dual Bcl-2/Bcl-xL inhibitor ABT-263 (navitoclax) greatly enhanced the activity of both compounds. These findings demonstrate that pharmacologic inhibition of Mcl-1 as a single agent or in combination with other cancer therapeutic agents is an effective way to modulate the intrinsic apoptotic pathway and promote cell death in cancer cells. Citation Format: John L. Sensintaffar, Allison Arnold, Craig Goodwin, Leah Hogdal, Subrata Shaw, James C. Tarr, Taekyu Lee, Edward Olejniczak, Stephen W. Fesik. Small molecule Mcl-1 inhibitors induce apoptosis and death in multiple cancer subtypes in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3727.

Diminished apoptotic priming and ATM signalling confer a survival advantage onto aged haematopoietic stem cells in response to DNA damage

Ageing of haematopoietic stem cells (HSCs) contributes to deficits in the aged haematopoietic system. HSC decline is driven in part by DNA damage accumulation; yet, how ageing impacts the acute DNA damage response (DDR) of HSCs is poorly understood. We show that old HSCs exhibit diminished ATM activity and attenuated DDR, leading to elevated clonal survival in response to a range of genotoxins that was underwritten by diminished apoptotic priming. Distinct HSC subsets exhibited ageing-dependent and subtype-dependent differences in apoptotic priming and survival in response to DNA damage. The defective DDR of old HSCs was non-cell autonomous, as ATM signalling and clonal survival in response to DNA damage could be restored to levels observed in young HSCs post-transplantated into young recipients. These data indicate that defective DDR and diminished apoptotic priming provide a selective advantage to old HSCs that may contribute to mutation accrual and disease predisposition.Gutierrez-Martinez et al. show that an impaired DNA damage response and reduced apoptotic priming in old haematopoietic stem cells (HSCs) contribute to the survival and expansion of damaged HSCs in the bone marrow of aged mice.