BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.
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 nM, and
cell death occurred within 2 h. Our ex vivo sensitivity results
compare favorably with those observed for chronic lymphocytic leukemia (CLL), 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.
Phosphorylation of Bcl2 at serine 70 may result from activation of a classic protein kinase C (PKC) isoform and is required for functional suppression of apoptosis by Bcl2 in murine growth factor-dependent cell lines (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). Human pre-B REH cells express high levels of Bcl2 yet remain sensitive to the chemotherapeutic agents etoposide, cytosine arabinoside, and Adriamycin. In contrast, myeloid leukemiaderived HL60 cells express less than half the level of Bcl-2 but are >10-fold more resistant to apoptosis induced by these drugs. The mechanism responsible for this apparent dichotomy appears to involve a deficiency of mitochondrial PKC␣ since 1) HL60 but not REH cells contain highly phosphorylated Bcl2; 2) PKC␣ is the only classical isoform co-localized with Bcl2 in HL60 but not REH mitochondrial membranes; 3) the natural product and potent PKC activator bryostatin-1 induces mitochondrial localization of PKC␣ in association with Bcl2 phosphorylation and increased REH cell resistance to drug-induced apoptosis; 4) PKC␣ can directly phosphorylate wild-type but not phosphorylation-negative and loss of function S70A Bcl2 in vitro; 5) stable, forced expression of exogenous PKC␣ induces mitochondrial localization of PKC␣, increased Bcl2 phosphorylation and a >10-fold increase in resistance to drug-induced cell death; and (6) PKC␣-transduced cells remain highly sensitive to staurosporine, a potent PKC inhibitor. Furthermore, treatment of the PKC␣ transformants with bryostatin-1 leads to even higher levels of mitochondrial PKC␣, Bcl2 phosphorylation, and REH cell survival following chemotherapy. While these findings strongly support a role for PKC␣ as a functional Bcl2 kinase that can enhance cell resistance to antileukemic chemotherapy, they do not exclude the possibility that another Bcl2 kinase(s) may also exist. Collectively, these findings identify a functional role for PKC␣ in Bcl2 phosphorylation and in resistance to chemotherapy and suggest a novel target for antileukemic strategies.
). The potent apoptotic agent ceramide can activate a PP2A, suggesting that one potential component of the ceramide-induced death signal may involve the inactivation of Bcl2. Results indicate that C2-ceramide but not inactive C2-dihydroceramide, was found to specifically activate a mitochondrial PP2A, which rapidly and completely induced Bcl2 dephosphorylation and correlated closely with ceramide-induced cell death. Using a genetic approach, the gain-of-function S70E Bcl2 mutation, which mimics phosphorylation, fails to undergo apoptosis even with the addition of high doses of ceramide (IC 50 > 50 M). In contrast, cells overexpressing exogenous wild-type Bcl2 were sensitive to ceramide at dosages where PP2A is fully active and Bcl2 would be expected to be dephosphorylated (IC 50 ؍ 14 M). These findings indicate that in cells expressing functional Bcl2, the mechanism of death action for ceramide may involve, at least in part, a mitochondrial PP2A that dephosphorylates and inactivates Bcl2.
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.
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