Targeted therapies are frequently combined with standard cytotoxic drugs to enhance clinical response. Targeting the B-cell lymphoma 2 (BCL-2) family of proteins is an attractive option to combat chemoresistance in leukemia. Preclinical and clinical studies indicate modest single-agent activity with selective BCL-2 inhibitors (for example, venetoclax). We show that venetoclax synergizes with cytarabine and idarubicin to increase antileukemic efficacy in a TP53-dependent manner. Although TP53 deficiency impaired sensitivity to combined venetoclax and chemotherapy, higher-dose idarubicin was able to suppress MCL1 and induce cell death independently of TP53. Consistent with an MCL1-specific effect, cell death from high-dose idarubicin was dependent on pro-apoptotic Bak. Combining higher-dose idarubicin with venetoclax was able to partially overcome resistance in Bak-deficient cells. Using inducible vectors and venetoclax to differentially target anti-apoptotic BCL-2 family members, BCL-2 and MCL1 emerged as critical and complementary proteins regulating cell survival in acute myeloid leukemia. Dual targeting of BCL-2 and MCL1, but not either alone, prolonged survival of leukemia-bearing mice. In conclusion, our findings support the further investigation of venetoclax in combination with standard chemotherapy, including intensified doses of idarubicin. Venetoclax should also be investigated in combination with direct inhibitors of MCL1 as a chemotherapy-free approach in the future.
Phosphoinositide signaling regulates diverse cellular functions. Phosphoinositide-3 kinase (PI3K) generates PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 , leading to the activation of proliferative and anti-apoptotic signaling pathways. Termination of phosphoinositide signaling requires hydrolysis of inositol ring phosphate groups through the actions of PtdIns(3,4,5)P 3 3-phosphatase (PTEN), PtdIns(3,4,5)P 3 5-phosphatases (eg, SHIP), and PtdIns(3,4)P 2 4-phosphatases (eg, INPP4B). The biological relevance of most of these phosphoinositide phosphatases in acute myeloid leukemia (AML) remains poorly understood. Mass spectrometry-based gene expression profiling of 3-, 4-and 5-phosphatases in human AML revealed significant overexpression of INPP4B. Analysis of an expanded panel of 205 AML cases at diagnosis revealed INPP4B overexpression in association with reduced responses to chemotherapy, early relapse, and poor overall survival, independent of other risk factors. Ectopic overexpression of INPP4B conferred leukemic resistance to cytosine arabinoside (ara-C), daunorubicin, and etoposide. Expression of a phosphatase inert variant (INPP4B C842A) failed to abrogate resistance of AML cells to chemotherapy in vitro or in vivo. In contrast, targeted suppression of endogenously overexpressed INPP4B by RNA interference sensitized AML cell lines and primary AML to chemotherapy. These findings demonstrate a previously unsuspected and clinically relevant role for INPP4B gain of function as a mediator of chemoresistance and poor survival outcome in AML independent of its phosphoinositide phosphatase function. (Blood. 2015;125(18):2815-2824
Summary Based on promising results in older adults with acute myeloid leukaemia (AML), we treated patients with NPM1mut measurable residual disease (MRD) using off‐label venetoclax in combination with low‐dose cytarabine or azacitidine. Twelve consecutive patients were retrospectively identified, including five with molecular persistence and seven with molecular relapse/progression. All patients with molecular persistence achieved durable molecular complete remission (CRMRD‐) without transplantation. Six of seven patients with molecular relapse/progression achieved CRMRD‐ after 1–2 cycles of venetoclax. This paper highlights the promising efficacy of venetoclax‐based therapy to reduce the relapse risk in patients with persistent or rising NPM1mut MRD.
Key Points Simultaneous inhibition of Cdk9 and PI3K in human AML cells liberates Bak from both Mcl-1 and Bcl-xL, inducing Bak-dependent apoptosis. Dual inhibitors of Cdk9 and PI3K, such as PIK-75, have broad activity against malignant cells including human AML cells.
Toxicity and drug resistance has impeded clinical improvements in acute myeloid leukaemia (AML). We hypothesized that directly targeting pro-survival proteins with BH3-mimetics may have therapeutic rationale in AML and potentially sensitize chemoresistant cases to chemotherapy. This work will therefore assess which Bcl-2 family members mediate pro-survival activity in AML and the potential benefit of combining BH3-mimetics with standard AML drugs. Immunoblotting of fresh primary AML samples revealed strong Bcl-2 expression in 12/13 (92%) cases, Bcl-xL expression in 3/13 (23%) cases and Mcl-1 expression in 8/13 cases (62%). Sensitivity to ABT-199 (targets Bcl-2) or ABT-737 (targets Bcl-2, -xL, -w) was assessed by incubation of freshly harvested AML samples in RPMI + 20% FBS for 48 hrs and viable cells enumerated by exclusion of Sytox Blue using flow cytometry. There were 2 discrete populations identified; one that was sensitive to ABT-199 with an LC50 (concentration of drug to kill 50% of AML blasts) < 10nM (5/13 cases; 39%) and a more resistant population (LC50 > 10μM in 6/13 cases, 46%). A strong correlation in the sensitivity of AML cells to ABT-199 and ABT-737 was observed (r=0.9628, p < 0.0001), suggesting Bcl-xL was not a dominant survival factor in AML. To identify relevant Bcl-2 pro-survival targets for elimination of AML in vivo, MV4;11 cells were engineered to express under doxycycline control 1) BimSwt targeting all pro-survival proteins, 2) BimS2A targeting Mcl-1 only, 3) BimSBad targeting Bcl-2, -x and –w; or 4) an inert BimS4E, which served as a negative control. 1x 105 MV4;11 cells per mouse were xenografted into cohorts of 6 NSG mice and doxycycline-rich water introduced on day 5 after transplantation. Mice transplanted with MV4;11 cells expressing the inert BimS4E succumbed to leukemia by ~ day 40. Expression of BimSBad or BimS2A delayed, but did not prevent leukemia-related death, whereas mice engrafted with MV4;11 cells enforced to express BimSwt were alive and leukemia-free after 100 days (Figure 1A). Similar outcomes were demonstrated using an OCI-AML xenograft model (not shown). As on-target toxicity to platelets is an undesirable consequence of targeting Bcl-xL (Mason, Cell 2006), the effectiveness of targeting only Bcl-2 and Mcl-1 in vivo was next investigated. Mice engrafted with MV4;11 cells expressing BimS2A (to target Mcl-1) were fed doxycycline water from day 5 onwards. In addition, the selective Bcl-2 inhibitor ABT-199 75mg/kg was administered daily by oral gavage between days 5-12 post engraftment. Strikingly, this combination strategy led to eradication of AML by day 100 in all mice, in contrast to delayed, but inevitable leukemic death in most mice receiving treatment directed at either Bcl-2 (ABT-199) or Mcl-1 (Bims2A) alone (Figure 1B). These results suggest that AML eradication in vivo is best achieved through combined targeting of Bcl-2 and Mcl-1. In the absence of direct inhibitors of Mcl-1, standard AML drugs were examined for their capacity to suppress Mcl-1. In the MV4;11 cell line, anthracyclines such as idarubicin rapidly (< 3 hrs) suppressed Mcl-1 at concentrations between 300nM and 1μM. In contrast, 100μM cytarabine did not suppress Mcl-1 after exposure for 6 hrs. Further studies in primary AML cells demonstrated synergy between ABT-199 and idarubicin in ABT-199 resistant AML cells. We conclude that 1) a subset of primary AML cells is highly sensitive to Bcl-2 inhibition, 2) that eradication of AML in vivo is best through combined Bcl-2/Mcl-1 targeting and 3) that anthracyclines are effective at suppressing Mcl-1 and could be combined with ABT-199 to treat patients with AML resistant to Bcl-2 targeted therapy. A Figure 1 A) Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS ligand variants under doxycycline control. Doxycycline water was fed to mice from day 5+ and survival assessed. B. Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS2A under doxycycline control. Mice were allocated to treatment with either vehicle or ABT-199 on days 5-12 +/- doxycycline water from day 5+ and survival assessed. All mice in the arm combining Doxycycline (to suppress Mcl-1) and ABT-199 (to target Bcl-2) were alive and leukemia-free on day 100. Figure 1. A) Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS ligand variants under doxycycline control. Doxycycline water was fed to mice from day 5+ and survival assessed. B. Cohorts of 6 NSG mice were xenografted with MV4;11 cells expressing BimS2A under doxycycline control. Mice were allocated to treatment with either vehicle or ABT-199 on days 5-12 +/- doxycycline water from day 5+ and survival assessed. All mice in the arm combining Doxycycline (to suppress Mcl-1) and ABT-199 (to target Bcl-2) were alive and leukemia-free on day 100. B Figure 2 Figure 2. Disclosures Huang: AbbVie: Consultancy, Honoraria. Wei:Abbvie: Consultancy, Honoraria.
Background: Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis leading to cytopenias, including anemia and thrombocytopenia. KER-050, a modified activin receptor type IIA inhibitor, is designed to target transforming growth factor-β ligands, including activin A. In preclinical studies, KER-050 promoted the maturation of progenitors across the full spectrum of erythropoiesis and thrombopoiesis and elicited bone anabolic effects. In a Phase 1 study in healthy participants, KER-050 treatment resulted in robust and sustained increases in reticulocytes (RETs), hemoglobin (HGB), and platelets. Increases in the bone formation marker bone specific alkaline phosphatase were also observed. Here we report results of an ongoing Phase 2 study to evaluate whether KER-050 provides therapeutic benefit in MDS patients with anemia. Aims: Evaluate safety, tolerability, pharmacodynamics and efficacy of ascending doses of KER-050 in participants with MDS in an open-label, 2-part Phase 2 study. Methods: IPSS-R very low-to-intermediate risk MDS patients (both RS+ and non-RS) with anemia (HGB <10g/dL or requiring RBC transfusions) are enrolled. In Part 1, ascending dose cohorts receive KER-050 subcutaneously every 4 weeks for 4 doses starting at 0.75mg/kg until a recommended Part 2 dose is determined. Part 2 dose expansion will begin following Part 1, with treatment extended to 2 years. Safety endpoints include incidence of adverse events (AEs); erythroid efficacy endpoints (≥8 weeks duration) include rates of transfusion independence (TI) in transfused participants, reduction in RBC transfusions by ≥4 units or ≥50% reduction in high transfusion burden participants (HTB) and HGB increase ≥1.5g/dL in non-transfused (NT) and low transfusion burden (LTB) participants. Results are reported for efficacy-evaluable participants in cohorts 1 and 2 of Part 1 dose escalation, defined as having ≥8 weeks of HGB and transfusion data. Results: At data cut-off (July 10, 2021) with median follow-up of 140 days (range 1 to 169 days), 17 participants had received ≥1 dose of KER-050 across 3 dose levels: 0.75 mg/kg, 1.5 mg/kg and 2.5 mg/kg. Baseline characteristics are described in Table 1. No related serious AEs, dose-limiting toxicities, or dose modifications were reported. One participant developed grade 2 maculopapular rash after the first dose which was considered treatment related, resolved and did not recur with subsequent doses. No other related AEs were reported. Two discontinued study drug prior to end of treatment: 1 due to participant decision, 1 due to death unrelated to study drug. None developed high risk MDS or AML. In 10 efficacy-evaluable participants, overall erythroid response rate was 60% (n=6/10). 33% (n=1/3) NT participants had a HGB increase of ≥1.5g/dL sustained ≥ 8 weeks. 5 of 7 transfused participants (71%) (n=1/2 LTB and n=4/5 HTB; n=2/3 non-RS and n=3/4 RS+) had erythroid responses sustained ≥8 weeks (range 8-20 weeks, ongoing) and 57% (n=4/7) achieved TI (Figure 1, Panel A). Maximum increase from baseline in RETs observed in transfused responders (TR) (n=5) was 24.6 x10 9/L (mean), range 10.5- 41.6 x10 9/L from day 1-29 with increases in RETs seen after each dose (Panel B). Maximum reduction in serum ferritin in TR was 40.4% (mean), range 10-66%, and maximum increase in soluble transferrin receptor (sTfR) was 52.8% (mean), range 29.8-116.4%. Increases in platelets were observed in TR (Panel C). Mean baseline platelet count for TR was 234 x10 9/L (range 104-401 x10 9/L), and maximum increase from baseline was 130 x10 9/L (mean), range 32-235 x10 9/L. No participants required dose reduction due to thrombocytosis. Summary: Erythroid responses have been observed in RS+ and non-RS MDS patients including reduction in transfusion burden at the initial dose levels. Observed increases in RETs and sTfR and observed decreases in ferritin suggest that KER-050 treatment is potentially associated with increased erythropoiesis. Increases in platelets have been observed in TR. These data support the potential of KER-050 as a treatment for multilineage cytopenias in MDS by potentially targeting multiple stages of hematopoiesis. As of data cut-off, KER-050 has been well tolerated. Dose escalation is ongoing in this Phase 2 study of anemic patients with MDS; data from planned cohorts from Part 1 will be presented. Part 2 dose expansion phase is expected to initiate prior to the meeting. Figure 1 Figure 1. Disclosures Ross: Bristol Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Keros Therapeutics: Consultancy, Honoraria. Arbelaez: Amgen: Other: Travel, Accommodations, Expenses. Chee: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fong: AbbVie: Consultancy; Amgen: Consultancy; Astellas: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy; Novartis: Consultancy, Speakers Bureau; Phizer: Consultancy; Novotech: Honoraria; Specialised Therapeutics: Honoraria. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Wight: Jannsen: Honoraria, Other: Travel subsidies; Abbvie: Honoraria, Other: Travel subsidies. Rovaldi: Keros Therapeutics: Current equity holder in publicly-traded company. Furutani: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaggi: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Jiang: Keros Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lachey: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Natarajan: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ordonez: Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees.
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