Rongqing Pan

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.

Inhibition of Mcl-1 with the pan-Bcl-2 family inhibitor (-)BI97D6 overcomes ABT-737 resistance in acute myeloid leukemia.

Overexpression of antiapoptotic Bcl-2 proteins such as Bcl-2, Bcl-xL, and Mcl-1 is widely associated with tumor initiation, progression, and chemoresistance. Furthermore, it has been demonstrated that Mcl-1 upregulation renders several types of cancers resistant to the Bcl-2/Bcl-xL inhibitors ABT-737 and ABT-263. The emerging importance of Mcl-1 in pathogenesis and drug resistance makes it a high-priority therapeutic target. In this study, we showed that inhibition of Mcl-1 with a novel pan-Bcl-2 inhibitor (-)BI97D6 potently induced apoptosis in acute myeloid leukemia (AML) cells. (-)BI97D6 induced hallmarks of mitochondrial apoptosis, disrupted Mcl-1/Bim and Bcl-2/Bax interactions, and stimulated cell death via the Bak/Bax-dependent mitochondrial apoptosis pathway, suggesting on-target mechanisms. As a single agent, this pan-Bcl-2 inhibitor effectively overcame AML cell apoptosis resistance mediated by Mcl-1 or by interactions with bone marrow mesenchymal stromal cells. (-)BI97D6 was also potent in killing refractory primary AML cells. Importantly, (-)BI97D6 killed AML leukemia stem/progenitor cells while largely sparing normal hematopoietic stem/progenitor cells. These findings demonstrate that pan-Bcl-2 inhibition by an Mcl-1-targeting inhibitor not only overcomes intrinsic drug resistance ensuing from functional redundancy of Bcl-2 proteins, but also abrogates extrinsic resistance caused by the protective tumor microenvironment.

The protein phosphatase 2A regulatory subunit B55α is a modulator of signaling and microRNA expression in acute myeloid leukemia cells

We recently discovered that the protein phosphatase 2A (PP2A) B55α subunit (PPP2R2A) is under-expressed in primary blast cells and is unfavorable for remission duration in AML patients. In this study, reverse phase protein analysis (RPPA) of 230 proteins in 511 AML patient samples revealed a strong correlation of B55α with a number of proteins including MYC, PKC α, and SRC. B55α suppression in OCI-AML3 cells by shRNA demonstrated that the B subunit is a PKCα phosphatase. B55α does not target SRC, but rather the kinase suppresses protein expression of the B subunit. Finally, the correlation between B55α and MYC levels reflected a complex stoichiometric competition between B subunits. Loss of B55α in OCI-AML3 cells did not change global PP2A activity and the only isoform that is induced is the one containing B56α. In cells containing B55α shRNA, MYC was suppressed with concomitant induction of the competing B subunit B56α (PPP2R5A). A recent study determined that FTY-720, a drug whose action involves the activation of PP2A, resulted in the induction of B55α In AML cells, and a reduction of the B subunit rendered these cells resistant to FTY-720. Finally, reduction of the B subunit resulted in an increase in the expression of miR-191-5p and a suppression of miR-142-3p. B55α regulation of these miRs was intriguing as high levels of miR-191 portend poor survival in AML, and miR-142-3p is mutated in 2% of AML patient samples. In summary, the suppression of B55α activates signaling pathways that could support leukemia cell survival.

Synthetic Lethality of Combined Bcl-2 Inhibition and p53 Activation in AML: Mechanisms and Superior Antileukemic Efficacy

Evasion of apoptosis is a hallmark of cancer. Bcl-2 and p53 represent two important nodes in apoptosis signaling pathways. We find that concomitant p53 activation and Bcl-2 inhibition overcome apoptosis resistance and markedly prolong survival in three mouse models of resistant acute myeloid leukemia (AML). Mechanistically, p53 activation negatively regulates the Ras/Raf/MEK/ERK pathway and activates GSK3 to modulate Mcl-1 phosphorylation and promote its degradation, thus overcoming AML resistance to Bcl-2 inhibition. Moreover, Bcl-2 inhibition reciprocally overcomes apoptosis resistance to p53 activation by switching cellular response from G1 arrest to apoptosis. The efficacy, together with the mechanistic findings, reveals the potential of simultaneously targeting these two apoptosis regulators and provides a rational basis for clinical testing of this therapeutic approach.

Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport

Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax.

Antileukemia efficacy and mechanisms of action of SL-101, a novel anti-CD123 antibody conjugate, in acute myeloid leukemia

Purpose: The persistence of leukemia stem cells (LSC)-containing cells after induction therapy may contribute to minimal residual disease (MRD) and relapse in acute myeloid leukemia (AML). We investigated the clinical relevance of CD34+CD123+ LSC-containing cells and antileukemia potency of a novel antibody conjugate SL-101 in targeting CD123+ LSCs. Experimental Methods and Results: In a retrospective study on 86 newly diagnosed AML patients, we demonstrated that a higher proportion of CD34+CD123+ LSC-containing cells in remission was associated with persistent MRD and predicted shorter relapse-free survival in patients with poor-risk cytogenetics. Using flow cytometry, we explored the potential benefit of therapeutic targeting of CD34+CD38−CD123+ cells by SL-101, a novel antibody conjugate comprising an anti-CD123 single-chain Fv fused to Pseudomonas exotoxin A. The antileukemia potency of SL-101 was determined by the expression levels of CD123 antigen in a panel of AML cell lines. Colony-forming assay established that SL-101 strongly and selectively suppressed the function of leukemic progenitors while sparing normal counterparts. The internalization, protein synthesis inhibition, and flow cytometry assays revealed the mechanisms underlying the cytotoxic activities of SL-101 involved rapid and efficient internalization of antibody, sustained inhibition of protein synthesis, induction of apoptosis, and blockade of IL3-induced p-STAT5 and p-AKT signaling pathways. In a patient-derived xenograft model using NSG mice, the repopulating capacity of LSCs pretreated with SL-101 in vitro was significantly impaired. Conclusions: Our data define the mechanisms by which SL-101 targets AML and warrant further investigation of the clinical application of SL-101 and other CD123-targeting strategies in AML. Clin Cancer Res; 23(13); 3385–95. ©2017 AACR.

Disruption of Wnt/β-catenin exerts anti-leukemia activity and synergizes with FLT3 inhibition in FLT3-mutant acute myeloid leukemia

Purpose: Wnt/β-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases β-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3-mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/β-catenin and FLT3 inhibition in FLT3-mutant AML. Experimental Design: Wnt/β-catenin signaling was inhibited by the β-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF. Results: We found significantly higher β-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow–resident leukemic cells compared with circulating blasts. Disrupting Wnt/β-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3-mutated AML cells and stem/progenitor cells in vitro. The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/β-catenin and FLT3 cooperatively decreased nuclear β-catenin and the levels of c-Myc and other Wnt/β-catenin and FLT3 signaling proteins. Importantly, β-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells. Conclusions: Disrupting Wnt/β-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3-mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3-mutated AML patients. Clin Cancer Res; 24(10); 2417–29. ©2018 AACR.

Abstract 605: Apogossypolone derivative -BI97D6 effectively targets MCL1 overexpressing Acute Myeloid Leukemia cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Aberrant expression of anti-apoptotic proteins such as BCL2, MCL1, and BCL-XL is observed in many tumors including Acute Myeloid Leukemia (AML). An obvious strategy for AML therapy would be to target the anti-apoptotic BCL2 family members. While compounds such as ABT-737 have shown promise for treatment of some malignancies, the compound binds poorly to MCL1 and thus cells that overexpress MCL1 usually display high resistance to the drug. Furthermore, ABT-737 activates ERK signaling resulting in MCL1 overexpression in surviving cells (Konopleva, et al, Leukemia 2011). The apogossypolone derivative -BI97D6 binds MCL1 and BCL2, BCL-XL, BFL1 with high affinities (Wei, et al, J Med Chem 2010). We recently reported that -BI97D6 (a) efficiently kills AML cell lines even in the presence of protective mesenchymal stroma cells, (b) interferes with the association between MCL1/BIM and BCL2/BAX, (c) stimulates BAX conformational change, (d) requires BAX or BAK for cell killing, and (e) does not induce ER stress. These data suggests that -BI97D6, unlike other gossypol or apogossypol compounds, acts mainly as a BH3 mimetic. In the current study, we first examined the role of MCL1 in -BI97D6-mediated killing. OCI-AML3 cells are extremely resistant to ABT-737 due to its high expression of MCL1. MCL1 expression was suppressed by lenti-viral shRNA in OCI-AML3 cells and knockdown of MCL1 protein by 80% was achieved as determined by quantitative western blot. Compared to cells with non-specific control shRNA, cells with MCL1 shRNA were greatly sensitized to ABT-737, and significantly sensitized to AraC, while -BI97D6, which binds MCL1 with high affinity, is almost equipotent in knockdown and control cells. Since results with AML cell lines have been promising, we examined the efficacy of -BI97D6 on primary AML samples. Flow cytometry analysis revealed that -BI97D6 potently induced apoptosis in AML blast cells at low nM concentration (EC50<50nM). -BI97D6 also effectively killed putative CD34+CD38- leukemia stem cells (LSC). Importantly, primary AML with FLT3-ITD and FLT3-D835 mutations showed similar sensitivity as those without FLT3 mutations (EC50<50nM). As FLT3 mutations are associated with poor prognosis in AML, these findings suggests that -BI97D6 can target AML LSC and could benefit even high risk patients. In summary, compound -BI97D6 may be a promising drug candidate for MCL1-targeted AML therapy. Citation Format: Rongqing Pan, Vivian Ruvolo, Maurizio Pellecchia, Jun Wei, Marina Konopleva, John C. Reed, Peter Ruvolo, Michael Andreeff. Apogossypolone derivative -BI97D6 effectively targets MCL1 overexpressing Acute Myeloid Leukemia cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2013-605

Abstract 2008: Broad spectrum BH3 mimetic BI-97D6 induces apoptosis of acute myeloid leukemia cells even when co-cultured with supporting stromal cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The BCL2 family of proteins contains members that possess potent anti-apoptotic function (e.g. BCL2, MCL-1) as well as pro-death members (e.g. BAX, BIM). Aberrant expression of anti-apoptotic members such as BCL2 or suppression of pro-apoptotic members such as BAX or BIM can lead to tumor formation and promote resistance to therapy in many types of cancer including Acute Myeloid Leukemia (AML). A recent model of tumorigenesis/chemoresistance suggests cancer cells become dependent on pro-survival molecules including BCL2 and MCL-1. This model suggests that the “addicted” malignant cells can be eliminated by targeting the survival molecule that supports the tumor cell. Recent efforts to target a broad group of BCL2 family members has evolved using small molecule inhibitors to target the BH3 domain since the association between the two groups of BCL2 family members involves this domain. While compounds including ABT-737 have shown promise for treatment of some cancers, the compound binds poorly to MCL-1 and related BCL2 family members such as BCL2A1 and thus cells that rely on these anti-apoptotic BCL2 family members display resistance to the compound. Furthermore, we and others have recently found that ABT-737 can promote MCL-1 expression likely via a mechanism involving ERK. A superior approach would be to develop a single BH-3 mimetic agent that is effective at inhibiting MCL-1 as well as other BCL2 family members. In the current study, we examine if the Apogossypol analog BI-97D6 (Wei et al, J Med Chem. 2010), which has efficacy against MCL-1 as well as BCL2 and BCL-XL, has potential an effective anti-leukemia agent. BI-97D6 is novel as an Apogossypol compound since it acts as a true BH3 mimetic. Other gossypol and apopgossypol compounds have been shown to promote death by ER stress mediated mechanisms. In AML cells, we found that BI-97D6 does not induce CHOP expression or JNK activation, which are features of ER stress induction. Bax/Bak knock out MEFs were resistant BI-97D6, indicating that the drug acts specifically as a BH3 mimetic. BI-97D6 interferes with the association of MCL-1 with BIM and induces the death conformation of BAX in OCI-AML3 cells. The drug is effective at killing a variety of AML derived cell lines with IC50 values at 72 hours in the nanomolar range (i.e. below 50 nM for OCI-AML3, HL60, and MOLM13). Bone marrow-derived mesenchymal stromal cells (BM MSC) have been shown to protect leukemia cells from conventional chemotherapy agents such as AraC. There was no difference in BI-97D6 mediated killing of OCI-AML3 cells whether the cells were treated in co-culture with BM MSC or alone. These results suggest that BI-97D6 could have efficacy against AML, regardless of MCL-1 levels, which is not diminished by the protective effects of the BM microenvironment. Further mechanistic details are under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2008. doi:1538-7445.AM2012-2008