Enzymes (PI3K and PTEN) controlling cellular levels of 3-phosphorylated phosphoinositides are known as important drivers or suppressors of tumorigenesis in various cancers. In this issue of Cancer Discovery, Kofuji and colleagues and Chew and colleagues identify the lipid phosphatase INPP4B as a context-specific tumor suppressor that controls phosphoinositide levels and AKT2 activation in PTEN-deficient cells.

Enzymes (PI3K and PTEN) controlling cellular levels of 3-phosphorylated phosphoinositides are known as important drivers or suppressors of tumorigenesis in various cancers. In this issue of Cancer Discovery, Kofuji and colleagues and Chew and colleagues identify the lipid phosphatase INPP4B as a context-specific tumor suppressor that controls phosphoinositide levels and AKT2 activation in PTEN-deficient cells.

Mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions as a key regulator of cell growth, division and survival. Many haematologic malignancies exhibit elevated or aberrant mTOR activation, supporting the launch of numerous clinical trials aimed at evaluating the potential of single agent mTOR-targeted therapies. While promising early clinical data using allosteric mTOR inhibitors (rapamycin and its derivatives, rapalogs) have suggested activity in a subset of haematologic malignancies, these agents have shown limited efficacy in most contexts. Whether the efficacy of these partial mTOR inhibitors might be enhanced by more complete target inhibition is being actively addressed with second generation ATP-competitive mTOR kinase inhibitors (TOR-KIs), which have only recently entered clinical trials. However, emerging preclinical data suggest that despite their biochemical advantage over rapalogs, TOR-KIs may retain a primarily cytostatic response. Rather, combinations of mTOR inhibition with other targeted therapies have demonstrated promising efficacy in several preclinical models. This review investigates the current status of rapalogs and TOR-KIs in B cell malignancies, with an emphasis on emerging preclinical evidence of synergistic combinations involving mTOR inhibition.

Abstract Targeting mTOR signaling is a promising approach for treating blood cancers. We reported that mTOR kinase inhibitors, including PP242 and MLN0128, synergize with ABL tyrosine kinase inhibitors (TKIs) to cause cell cycle arrest and death in acute leukemia cells driven by BCR-ABL. mTOR kinase inhibitors are more effective than rapamycin in these models and have minimal effects on normal hematopoietic cells and immune responses at anti-leukemic doses. Ongoing studies indicate that mTOR kinase inhibitors are immunosuppressive at slightly higher concentrations. Moreover, the compounds are generally not cytotoxic as single agents in leukemia or lymphoma models. These findings emphasize the need for rational combinations to unleash the therapeutic potential of mTOR kinase inhibitors. To this end, we have tested various classes of anti-cancer agents guided by gene expression and proteomic data. Our data reveal synergies between mTOR kinase inhibitors with histone deacetylase inhibitors in B-ALL, and with BCL2 antagonists in DLBCL. Unexpectedly, mTOR kinase inhibitors protect B-ALL cells from methotrexate and 6-mercaptopurine, chemotherapeutic agents used in the treatment of B-ALL patients. ABL TKIs can also protect B-ALL cells from methotrexate by inhibiting downstream mTOR signaling. Together these studies identify potential applications and limitations of mTOR-targeted therapy in blood cancers. Citation Format: Thanh-Trang Vo, Jong-Hoon Scott Lee, Lomon So, Brandon Beagle, Matthew R. Janes, David A. Fruman. Mechanisms of resistance to mTOR inhibitors in leukemia and lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr IA16.

The PI3K/AKT/mTOR axis promotes survival and is a frequently mutated pathway in cancer. Yet, inhibitors targeting this pathway are insufficient to induce cancer cell death as single agents in some contexts, including diffuse large B cell lymphoma (DLBCL). In these situations, combinations with inhibitors targeting BCL-2 survival proteins (ABT-199 and ABT-263) may hold potential. Indeed, studies have demonstrated marked synergy in contexts where PI3K/mTOR inhibitors suppress expression of the pro-survival protein, MCL-1. In this study, we use BH3 profiling to confirm that BCL-2 and BCL-XL support survival following PI3K pathway inhibition, and that the dual PI3K/mTOR inhibitor BEZ235 strongly synergizes with BCL-2 antagonists in DLBCL. However, we identify an alternative mechanism of synergy between PI3K/mTOR and BCL-2 inhibitors, independent of MCL-1 down-regulation. Instead, we show that suppression of AKT activation by BEZ235 can induce the mitochondrial accumulation of pro-apoptotic BAD and BIM, and that expression of a constitutively active form of AKT prevents sensitization to BCL-2 antagonism. Thus, our work identifies an additional mechanism of synergy between PI3K pathway inhibitors and BCL-2 antagonists that strengthens the rationale for testing this combination in DLBCL.

Abstract B-ALL is the most prominent cancer in pediatric patients. Most patients are responsive to the current chemotherapy, but B-ALLs with activated oncogenic kinases including BCR-ABL, PDGFR and JAK have a high risk of relapse. Currently, patients with the BCR-ABL translocation are successfully treated when the BCL-ABL inhibitor dasatinib is added to their chemotherapy regimen. In accord, we found that dasatinib sensitizes B-ALL cell lines to drugs used during induction and consolidation phase of chemotherapy. However, dasatinib protects BCR-ABL leukemia cell lines from killing by maintenance phase drugs methotrexate and 6-mercaptopurine. This protection only occurs in cells where dasatinib reduces downstream mTOR activity. To directly test if reduced mTOR activity can account for the protection, we assessed the effects of the allosteric mTOR inhibitor rapamycin and active-site mTOR inhibitor MLN0128. Indeed, both mTOR inhibitors consistently protect B-ALL cells from killing by methotrexate and 6-mercaptopurine. Inhibitors of AKT and PI3K also result in varying degrees of chemoprotection, which correlates strongly with the degree to which they downregulate mTOR activity. The chemoprotective effect of mTOR inhibition correlates with cell cycle arrest but other mechanisms may also be involved. These data suggest that mTOR inhibition is detrimental in some chemotherapeutic combinations, whether by inhibitors directly targeting mTOR or indirectly targeting upstream effectors. The identification of such drug combination effects is an important consideration for the current use of dasatinib and future use of PI3K/AKT/mTOR inhibitors. Citation Format: Thanh-Trang Vo, Duc Nguyen, Brandon Lui, Mengrou Lu, Sharmila Mallya, David Fruman. Inhibition of mTOR induces chemoresistance in B-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A49.

High-risk subtypes of B-cell acute lymphoblastic leukemia (B-ALL) include Philadelphia chromosome-positive (Ph+) B-ALL driven by the BCR-ABL1 oncogene and a more recently identified subtype known as BCR-ABL-like or Ph-like B-ALL. A hallmark of both Ph+ and Ph-like B-ALL is constitutive activation of tyrosine kinase signaling that is potentially targetable with tyrosine kinase inhibitors (TKIs). B-ALL cells also receive extracellular signals from the microenvironment that can maintain proliferation and survival following treatment with TKIs. Therefore, there is strong rationale for combining TKIs with other therapies targeting signal transduction pathways. Here we show that combinations of the ABL-directed TKI dasatinib with mTOR kinase inhibitors (TOR-KIs) are more effective than TKI alone against patient-derived Ph-like B-ALL cells harboring rearrangements of ABL1 or ABL2. We also report the establishment of a new human Ph-like B-ALL cell line that is stromal cell-independent in vitro and can be used for xenograft experiments in vivo. These findings provide rationale for clinical testing of TKI plus TOR-KIs in children and adults with Ph-like B-ALL and a new experimental tool to test promising therapeutic strategies in this poor prognosis subtype of B-ALL.

Elevated activity of mTOR is associated with poor prognosis and higher incidence of relapse in B-cell acute lymphoblastic leukemia (B-ALL). Thus, ongoing clinical trials are testing mTOR inhibitors in combination with chemotherapy in B-ALL. However, the combination of mTOR inhibitors with standard of care chemotherapy drugs has not been studied extensively in high-risk B-ALL subtypes. Therefore, we tested whether mTOR inhibition can augment the efficacy of current chemotherapy agents in Ph⁺ and Ph-like B-ALL models. Surprisingly, inhibiting mTOR complex 1 (mTORC1) protected B-ALL cells from killing by methotrexate and 6-mercaptopurine, two antimetabolite drugs used in maintenance chemotherapy. The cytoprotective effects correlated with decreased cell-cycle progression and were recapitulated using cell-cycle inhibitors, palbociclib or aphidicolin. Dasatinib, a tyrosine kinase inhibitor currently used in Ph⁺ patients, inhibits ABL kinase upstream of mTOR. Dasatinib resistance is mainly caused by ABL kinase mutations, but is also observed in a subset of ABL unmutated cases. We identified dasatinib-resistant Ph+ cell lines and patient samples in which dasatinib can effectively reduce ABL kinase activity and mTORC1 signaling without causing cell death. In these cases, dasatinib protected leukemia cells from killing by 6-mercaptopurine. Using xenograft models, we observed that mTOR inhibition or dasatinib increased the numbers of leukemia cells that emerge after cessation of chemotherapy treatment. These results demonstrate that inhibitors targeting mTOR or upstream signaling nodes should be used with caution when combined with chemotherapeutic agents that rely on cell-cycle progression to kill B-ALL cells. Mol Cancer Ther; 16(9); 1942-53. ©2017 AACR.

High activity of the mechanistic target of rapamycin (mTOR) is associated with poor prognosis in pre-B-cell acute lymphoblastic leukemia (B-ALL), suggesting that inhibiting mTOR might be clinically useful. However, emerging data indicate that mTOR inhibitors are most effective when combined with other target agents. One strategy is to combine with histone deacetylase (HDAC) inhibitors, since B-ALL is often characterized by epigenetic changes that silence the expression of pro-apoptotic factors. Here we tested combinations of mTOR and pan-HDAC inhibitors on B-ALL cells, including both Philadelphia chromosome-positive (Ph+) and non-Ph cell lines. We found that mTOR kinase inhibitors (TOR-KIs) synergize with HDAC inhibitors to cause apoptosis in B-ALL cells and the effect is greater when compared to rapamycin plus HDAC inhibitors. The combination of TOR-KIs with the clinically approved HDAC inhibitor vorinostat increased apoptosis in primary pediatric B-ALL cells in vitro. Mechanistically, TOR-KI and HDAC inhibitor combinations increased expression of pro-death genes, including targets of the Forkhead Box O (FOXO) transcription factors, and increased sensitivity to apoptotic triggers at the mitochondria. These findings suggest that targeting epigenetic factors can unmask the cytotoxic potential of TOR-KIs towards B-ALL cells.

The mechanistic target of rapamycin (mTOR) is a kinase whose activation is associated with poor prognosis in pre-B cell acute lymphoblastic leukemia (B-ALL). These and other findings have prompted diverse strategies for targeting mTOR signaling in B-ALL and other B-cell malignancies. In cellular models of Philadelphia Chromosome-positive (Ph+) B-ALL, mTOR kinase inhibitors (TOR-KIs) that inhibit both mTOR-complex-1 (mTORC1) and mTOR-complex-2 (mTORC2) enhance the cytotoxicity of tyrosine kinase inhibitors (TKIs) such as dasatinib. However, TOR-KIs have not shown substantial efficacy at tolerated doses in blood cancer clinical trials. Selective inhibition of mTORC1 or downstream effectors provides alternative strategies that may improve selectivity towards leukemia cells. Of particular interest is the eukaryotic initiation factor 4F (eIF4F) complex that mediates cap-dependent translation. Here we use novel chemical and genetic approaches to show that selective targeting of either mTORC1 kinase activity or components of the eIF4F complex sensitizes murine BCR-ABL-dependent pre-B leukemia cells to dasatinib. SBI-756, a small molecule inhibitor of eIF4F assembly, sensitizes human Ph+ and Ph-like B-ALL cells to dasatinib cytotoxicity without affecting survival of T lymphocytes or natural killer cells. These findings support the further evaluation of eIF4F-targeted molecules in combination therapies with TKIs in B-ALL and other blood cancers.