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Supplemental Table S1: Stromal Dependent PDX samples; Supplemental Figure S1: Protection by mTOR inhibition is dose dependent; Supplemental Figure S2: Effect of mTOR inhibition on other chemotherapies; Supplemental Figure S3: Titration of PI3K/AKT/mTOR inhibitors; Supplemental Figure S4: Dose of dasatinib used inhibits ABL activity in Ph+ lines; Supplemental Figure S5: Killing effect of dasatinib alone can counteract the protection by mTOR inhibition; Supplemental Figure S6: Palbociclib alone is not cytotoxic; Supplemental Figure S7: Cell cycle inhibition reduces DNA damage by maintenance drugs; Supplemental Figure S8: mTOR inhibition decreases DHFR expression in some PDX lines

Monocytes are among the first cells recruited to sites of infection and major producers of the potent proinflammatory cytokine IL-1β. We previously showed that IL-1β release duringToxoplasma gondiiinfection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 activity but is independent of gasdermin D and pyroptosis. To investigate potential mechanisms of pyroptosis-independent release of IL-1β duringT. gondiiinfection, we constructed caspases-1, -4, -5, or -8 knockout THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1β release duringT. gondiiinfection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 knockout cells duringT. gondiiinfection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1β release without affecting cell viability or parasite infection efficiency. In addition, caspase-8 was required for the release of active caspase-1 fromT. gondii-infected cells and for IL-1β release during infection with the related apicomplexan parasiteNeospora caninum. Surprisingly, caspase-8 was dispensable for the synthesis and cleavage of IL-1β, but caspase-8 deficiency resulted in theretentionof mature IL-1β within cells. Our data indicate that duringT. gondiiinfection of human monocytes, caspase-8 functions in a novel gasdermin D-independent mechanism controlling IL-1β release from viable cells. This study expands on the known molecular pathways that promote IL-1β in human immune cells and provides the first evidence of a role for caspase-8 in the mechanism of IL-1β release during host defense against infection.

Background The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). Methods We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. Results Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. Conclusions Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.

The mechanistic target of rapamycin (mTOR) is a kinase whose activity is elevated in hematological malignancies. mTOR-complex-1 (mTORC1) phosphorylates numerous substrates to promote cell proliferation and survival. Eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs) are mTORC1 substrates with an integral role in oncogenic protein translation. Current pharmacological approaches to inhibit mTORC1 activity and 4E-BP phosphorylation have drawbacks. Recently we described a series of bi-steric compounds that are potent and selective inhibitors of mTORC1, inhibiting 4E-BP phosphorylation at lower concentrations than mTOR kinase inhibitors (TOR-KIs). Here we report the activity of the mTORC1-selective bi-steric inhibitor, RMC-4627, in BCR-ABL-driven models of B-cell acute lymphoblastic leukemia (B-ALL). RMC-4627 exhibited potent and selective inhibition of 4E-BP1 phosphorylation in B-ALL cell lines without inhibiting mTOR-complex-2 (mTORC2) activity. RMC-4627 suppressed cell cycle progression, reduced survival, and enhanced dasatinib cytotoxicity. Compared to a TOR-KI compound, RMC-4627 was more potent, and its effects on cell viability were sustained after washout in vitro. Notably, a once-weekly, well tolerated dose reduced leukemic burden in a B-ALL xenograft model and enhanced the activity of dasatinib. These preclinical studies suggest that intermittent dosing of a bi-steric mTORC1-selective inhibitor has therapeutic potential as a component of leukemia regimens, and further study is warranted.

Summary The cap-binding protein eukaryotic initiation factor 4E (eIF4E) promotes translation of mRNAs associated with proliferation and survival and is an attractive target for cancer therapeutics. Here, we used Eif4e germline and conditional knockout models to assess the impact of reduced Eif4e gene dosage on B-cell leukemogenesis compared to effects on normal pre-B and mature B-cell function. Using a BCR-ABL-driven pre-B-cell leukemia model, we find that loss of one allele of Eif4e impairs transformation and reduces fitness in competition assays in vitro and in vivo. In contrast, reduced Eif4e gene dosage had no significant effect on development of pre-B and mature B cells or on survival or proliferation of non-transformed B lineage cells. These results demonstrate that inhibition of eIF4E could be a new therapeutic tool for pre-B-cell leukemia while preserving development and function of normal B cells., Graphical abstract, Highlights • Loss of one allele of Eif4e impairs pre-B-cell leukemia transformation • Eif4e+/- leukemia cells had reduced competitive fitness both in vitro and in vivo • Reduced Eif4e dosage had no effect on B-cell development, survival, or proliferation, Molecular biology; Cell biology; Cancer

Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis., Toxoplasma gondii can infect and replicate in vascular endothelial cells prior to entering host tissues. However, little is known about the molecular interactions at the parasite-endothelial cell interface. We demonstrate that T. gondii infection of primary human umbilical vein endothelial cells (HUVEC) altered cell morphology and dysregulated barrier function, increasing permeability to low-molecular-weight polymers. T. gondii disrupted vascular endothelial cadherin (VE-cadherin) and β-catenin localization to the cell periphery and reduced VE-cadherin protein expression. Notably, T. gondii infection led to reorganization of the host cytoskeleton by reducing filamentous actin (F-actin) stress fiber abundance under static and microfluidic shear stress conditions and by reducing planar cell polarity. RNA sequencing (RNA-Seq) comparing genome-wide transcriptional profiles of infected to uninfected endothelial cells revealed changes in gene expression associated with cell-cell adhesion, extracellular matrix reorganization, and cytokine-mediated signaling. In particular, genes downstream of Hippo signaling and the biomechanical sensor and transcriptional coactivator Yes-associated protein (YAP) were downregulated in infected endothelial cells. Interestingly, T. gondii infection activated Hippo signaling by increasing phosphorylation of LATS1, leading to cytoplasmic retention of YAP, and reducing YAP target gene expression. These findings suggest that T. gondii infection triggers Hippo signaling and YAP nuclear export, leading to an altered transcriptional profile of infected endothelial cells. IMPORTANCE Toxoplasma gondii is a foodborne parasite that infects virtually all warm-blooded animals and can cause severe disease in individuals with compromised or weakened immune systems. During dissemination in its infected hosts, T. gondii breaches endothelial barriers to enter tissues and establish the chronic infections underlying the most severe manifestations of toxoplasmosis. The research presented here examines how T. gondii infection of primary human endothelial cells induces changes in cell morphology, barrier function, gene expression, and mechanotransduction signaling under static conditions and under the physiological conditions of shear stress found in the bloodstream. Understanding the molecular interactions occurring at the interface between endothelial cells and T. gondii may provide insights into processes linked to parasite dissemination and pathogenesis.

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection., Author summary IL-1β is a proinflammatory cytokine that contributes to host defense against infection and is also associated with autoimmune and inflammatory diseases. Our prior research has demonstrated that the intracellular parasite Toxoplasma gondii induces IL-1β release from primary human monocytes during infection. Here we report the novel finding that within minutes of infection, T. gondii activates a spleen tyrosine kinase (Syk), PKCδ, CARD9/MALT-1, and NF-κB signaling pathway that is critical for the production of IL-1β in primary human monocytes. We have also investigated the mechanism of IL-1β release from monocytes. Interestingly, although IL-1β can be released during pyroptotic cell death, which is driven by gasdermin family proteins such as gasdermin D (GSDMD), we have found that T. gondii triggers the release of IL-1β from viable cells, independent of GSDMD, thereby preserving the parasite’s intracellular niche. These studies provide mechanistic insight into the regulation of inflammation and host defense against parasite infection by human innate immune cells.

mTORC1 signaling is elevated in most lymphoid malignancies and frequently is associated with poor prognosis. Targeting mTORC1 with rapalogs or mTOR kinase inhibitors (TOR-KIs) are two strategies that both have limitations. Rapalogs are weak inducers of apoptosis, mainly due to incomplete mTORC1 inhibition, resulting in reduced phosphorylation of some substrates such as S6K1, but not 4E-BP1. TOR-KIs inhibit mTORC1 more completely, but also inhibit mTORC2, which likely contributes to dose-limiting toxicities in TOR-KI clinical trials. We have evaluated alternative strategies to target mTORC1 and downstream survival pathways in B-cell leukemia and lymphoma. In one approach, we have used novel bi-steric inhibitors of mTORC1, generated using our RevBlocksTM modular synthesis platform. These inhibitors engage both the FRB- and ATP-binding sites of mTOR and exhibit potent and selective inhibition of mTORC1. We found that RM-001, a proprietary bi-steric compound ~13-fold-selective for mTORC1 over mTORC2 in vitro, enhances the efficacy of the BCR-ABL1 inhibitor dasatinib in Philadelphia chromosome-positive pre-B cell acute lymphoblastic leukemia (Ph+ B-ALL) cell lines, causing profound and sustained phosphorylation inhibition of 4E-BP1 and S6, cell growth inhibition, and cell cycle arrest. Once-weekly dosing with RM-001 (10 mg/kg, IP) in a Ph+ B-ALL mouse model was well tolerated and effectively reduced leukemic burden as a single agent. A corresponding inhibition of phosphorylation of 4E-BP1 and S6 was observed in bone marrow. A key downstream effector pathway of mTORC1 is formation of the eIF4F translation initiation complex, which consists of eIF4E, eIF4G, and eIF4A. To assess targeting eIF4F as an alternative approach, we evaluated the function of eIF4F in both tumors and normal cells of the B lymphocyte lineage. In both B-ALL and diffuse large B-cell lymphoma (DLBCL) cells, disruption of eIF4F with a constitutively active 4E-BP1 mutant phenocopied the effect of TOR-KIs and bi-steric inhibitors. Notably, reduced gene dosage of Eif4e (eIF4E+/-) impaired the initiation and maintenance of B-cell transformation without affecting normal B-cell development and function. Most existing chemical inhibitors of eIF4F target either the cap-binding protein eIF4E or the RNA helicase eIF4A. We tested a novel eIF4G-binding molecule, SBI-756, shown previously to overcome resistance to BRAF inhibitors in melanoma models. SBI-756 (250 – 500 nM) disrupted eIF4F formation and caused marked cytotoxic effects in B-ALL, DLBCL, and normal B lymphocytes, while sparing T cells and NK cells. SBI-756 also synergized with targeted agents including dasatinib in Ph+ B-ALL and venetoclax in DLBCL. The combination of SBI-756 with venetoclax was more effective than single agents in a DLBCL xenograft model. These findings establish proof of concept for molecules that selectively bind to the scaffolding protein eIF4G. Collectively, these data highlight distinct strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma. Citation Format: Lee-or Herzog, Bianca J. Lee, Thanh-Trang Vo, Honyin Chiu, Sharmila Mallya, Amos Fung, Mallika Singh, James Aggen, Nidhi Tibrewal, Jacqueline A.M. Smith, David Wildes, Ze'ev Ronai, Davide Ruggero, David A. Fruman. Strategies to target the mTORC1/eIF4F axis in B-cell leukemia and lymphoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr IA17.

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.

Statins have shown promise as anticancer agents in experimental and epidemiologic research. However, any benefit that they provide is likely context-dependent, for example, applicable only to certain cancers or in combination with specific anticancer drugs. We report that inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) using statins enhances the proapoptotic activity of the B cell lymphoma-2 (BCL2) inhibitor venetoclax (ABT-199) in primary leukemia and lymphoma cells but not in normal human peripheral blood mononuclear cells. By blocking mevalonate production, HMGCR inhibition suppressed protein geranylgeranylation, resulting in up-regulation of proapoptotic protein p53 up-regulated modulator of apoptosis (PUMA). In support of these findings, dynamic BH3 profiling confirmed that statins primed cells for apoptosis. Furthermore, in retrospective analyses of three clinical studies of chronic lymphocytic leukemia, background statin use was associated with enhanced response to venetoclax, as demonstrated by more frequent complete responses. Together, this work provides mechanistic justification and clinical evidence to warrant prospective clinical investigation of this combination in hematologic malignancies.

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.

Rapamycin has been used as a clinical immunosuppressant for many years; however, the molecular basis for its selective effects on lymphocytes remains unclear. We investigated the role of two canonical effectors of the mammalian target of rapamycin (mTOR): ribosomal S6 kinases (S6Ks) and eukaryotic initiation factor 4E (eIF4E)-binding proteins (4E-BPs). S6Ks are thought to regulate cell growth (increase in cell size), and 4E-BPs are thought to control proliferation (increase in cell number), with mTORC1 signaling serving to integrate these processes. However, we found that the 4E-BP-eIF4E signaling axis controlled both the growth and proliferation of lymphocytes, processes for which the S6Ks were dispensable. Furthermore, rapamycin disrupted eIF4E function selectively in lymphocytes, which was due to the increased abundance of 4E-BP2 relative to that of 4E-BP1 in these cells and the greater sensitivity of 4E-BP2 to rapamycin. Together, our findings suggest that the 4E-BP-eIF4E axis is uniquely rapamycin-sensitive in lymphocytes and that this axis promotes clonal expansion of these cells by coordinating growth and proliferation.

The purpose of this project is to investigate the feasibility of using small molecules targeting cap-dependent translation to sensitize B cell lymphoma cells to killing by BCL-2 inhibitors (venetoclax/ ABT-199). We hypothesized that diffuse large B-cell lymphoma (DLBCL) cell survival requires cap-dependent translation, which is facilitated by eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interaction and is promoted by mTOR complex 1 (mTORC1). We used the compound SBI-756, a novel inhibitor targeting the scaffolding protein eIF4G (1). SBI-756 is more potent and selective than previously reported compounds targeting the eIF4E cap-binding protein, or compounds targeting eIF4A helicase. By treating DLBCL cells (germinal center B-cell subtype) with SBI-756 (sub μM concentrations), we discovered profound synergistic induction of apoptosis when combined with venetoclax. Cell viability was reduced even more compared to mTOR kinase inhibitor (TOR-KI) treatment combination. Moreover, SBI-756 was found to reduce the viability (IC50 = 653nM) of VAL lymphoma cells lacking 4E-BP1, while VAL were insensitive to TOR-KI treatment. By using Proximity Ligation Assay (PLA) we showed that SBI-756 treatment prevents eIF4E-eIF4G association in intact lymphoma cells. Dual luciferase assays validated this and indicated a dose-dependent reduction in cap-dependent translation following SBI-756 treatment. Also, Western blot analyses confirmed that SBI-756 treatment did not change mTOR substrates' phosphorylation, indicating that the SBI-756 effect is specific to preventing the eIF4E-eIF4G interaction. Furthermore, SBI-756 treatment reduced polysome formation with a corresponding increase in abundance of monosomes. Lastly, induced expression of 4E-BP1 mutant, which irreversibly binds eIF4E, sensitized DLBCL cells to venetoclax treatment to a similar extent as TOR-KI treatment. Ongoing studies will investigate: (1) The molecular mechanism by which translation inhibition by SBI-756 synergizes with venetoclax to prime DLBCL cells toward apoptosis, and (2) pharmacokinetic and pharmacodynamic effects of venetoclax and SBI-756 treatment on DLBCL progression in vivo. Hence, this project highlights a novel combination for treatment of aggressive lymphomas, and establishes its efficacy using preclinical models. Reference: 1. Feng Y., et al. SBI-0640756 attenuates the growth of clinically unresponsive melanomas by disrupting the eIF4F translation initiation complex. Cancer Res 2015;75(24):OF1-8. Citation Format: Lee-or Herzog, Nancy Nguyen, Honyin Chiu, Sharmila Mallya, Ze'ev A. Ronai, David A. Fruman. A novel inhibitor of eIF4F protein translation complex sensitizes DLBCL cells to BCL-2 targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1886.

Phosphoinositide 3-kinases (PI3Ks) are promising targets for therapeutic development in cancer. The class I PI3K isoform p110α has received considerable attention in oncology because the gene encoding p110α (PIK3CA) is frequently mutated in human cancer. However, little is known about the function of p110α in lymphocyte populations that modulate tumorigenesis. We used recently developed investigational inhibitors to compare the function of p110α and other isoforms in natural killer (NK) cells, a key cell type for immunosurveillance and tumor immunotherapy. Inhibitors of all class I isoforms (pan-PI3K) significantly impaired NK cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity against tumor cells, whereas p110α-selective inhibitors had no effect. In NK cells stimulated through NKG2D, p110α inhibition modestly reduced PI3K signaling output as measured by AKT phosphorylation. Production of IFN-γ and NK cell-derived chemokines was blocked by a pan-PI3K inhibitor and partially reduced by a p110δinhibitor, with lesser effects of p110α inhibitors. Oral administration of mice with MLN1117, a p110α inhibitor in oncology clinical trials, had negligible effects on NK subset maturation or terminal subset commitment. Collectively, these results support the targeting of PIK3CA mutant tumors with selective p110α inhibitors to preserve NK cell function. © 2014 Yea et al.

Inhibitors of the mechanistic target of rapamycin (mTOR) hold promise for treatment of hematological malignancies. Analogs of the allosteric mTOR inhibitor rapamycin are approved for mantle cell lymphoma but have limited efficacy in other blood cancers. ATP-competitive "active-site" mTOR inhibitors produce more complete mTOR inhibition and are more effective than rapamycin in preclinical models of leukemia, lymphoma and multiple myeloma. In parallel to clinical trials of active-site mTOR inhibitors, it will be important to identify resistance mechanisms that might limit drug efficacy in certain patients. From a panel of diffuse large B-cell lymphoma cell lines, we found that the VAL cell line is particularly resistant to apoptosis in the presence of active-site mTOR inhibitors. Mechanistic investigation showed that VAL does not express eukaryotic initiation factor 4E-binding protein-1 (4EBP1), a key negative regulator of translation controlled by mTOR. Although VAL cells express the related protein 4EBP2, mTOR inhibitor treatment fails to displace eukaryotic initiation factor 4G from the mRNA cap-binding complex. Knockdown of eukaryotic initiation factor 4E, or re-expression of 4EBP1, sensitizes cells to apoptosis when treated with active-site mTOR inhibitors. These findings provide a naturally occurring example of 4EBP deficiency driving lymphoma cell resistance to active-site mTOR inhibitors.

Background and Rationale: B-lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. While event-free survival (EFS) exceeds 85% for most patients treated with contemporary therapy, outcomes are very poor for children who relapse, highlighting a need for new treatments. In particular, children with Philadelphia chromosome-like (Ph-like) B-ALL (who lack BCR-ABL1 rearrangement) have high rates of relapse and mortality with conventional chemotherapy. Transcriptional profiling and genomic sequencing of Ph-like ALL specimens have identified a variety of alterations that activate oncogenic kinase signaling, including rearrangements (R) of CRLF2, ABL1, and PDGFRB. Addition of the tyrosine kinase inhibitor (TKI) imatinib to chemotherapy has dramatically improved EFS for patients with BCR-ABL1-rearranged (Ph+) B-ALL, and it is hypothesized that TKI addition to therapy will similarly improve outcomes for patients with Ph-like ALL. Our prior preclinical studies in Ph+ B-ALL demonstrated enhanced efficacy of combining TKIs (imatinib or dasatinib) with mTOR kinase inhibitors (TOR-KIs) (Janes et al., Nature Medicine 2010; Janes et al, Leukemia2013). In the current studies, we hypothesized that dual kinase inhibitor therapy would have superior anti-leukemia cytotoxicity in Ph-like ALL and thus investigated combined TKI and TOR-KI treatment using patient-derived xenograft (PDX) models of childhood Ph-like ALL. Methods: For in vitro studies, viably cryopreserved leukemia cells from established ABL1-R Ph-like ALL PDX models (2 ETV6-ABL1) were incubated with the TKI dasatinib, TOR-KIs, or both TKI + TOR-KI for 72 hours prior to flow cytometric assessment of cellular viability via Annexin V and propidium iodide staining. Two chemically distinct TOR-KIs (MLN0128 or AZD2014) were used to confirm on-target effects. Additional primary ABL1-R or PDGFRB-R Ph-like ALL specimens were plated in methylcellulose without or with inhibitors in colony-forming assays. Phosphoflow cytometry (PFC) analysis of ALL cells incubated with inhibitors was also performed to measure the ability of TKIs and TOR-KIs to inhibit intracellular ABL1 and PI3K/mTOR signaling pathways. For in vivo studies, Ph-like ALL PDX models were treated with dasatinib, the TOR-KI AZD8055, or both drugs via daily oral gavage for 8 days. Human CD19+ ALL was quantified in murine spleens and bone marrow at end of treatment with quantification of cycling cells by EdU incorporation. PFC analysis of murine bone marrow was also performed 2 hours after drugs were dosed, to measure in vivo inhibition of signaling proteins. Results: Combined in vitro treatment with dasatinib and MLN0128 or AZD2014 decreased cellular viability more than inhibitor monotherapy. Similarly, in a set of CRLF2-rearranged samples, mTOR inhibitors augmented killing by the JAK2 inhibitor BBT-594. Incubation of primary ABL1-R or PDGFRB-R ALL cells with both dasatinib and AZD2014 more robustly inhibited colony formation than did inhibitor monotherapy. In in vitro PFC analyses of ABL1-R samples, we observed expected dasatinib-induced inhibition of phosphorylated (p) STAT5. Inhibition of the mTOR substrate pS6 was observed with dasatinib, MLN0128, and AZD2014 with more complete inhibition achieved when dasatinib combined with either MLN0128 or AZD2014. Similarly, in vivo treatment of PDX models with dasatinib and AZD8055 reduced leukemia burden and pS6 signaling more completely than either inhibitor alone. Importantly, dual inhibition decreased the percentage of cycling human ALL cells in murine bone marrow, but preserved cycling in normal mouse bone marrow cells in the same animals. Our data thus provide additional compelling preclinical rationale for combined inhibitor therapy with TKIs and TOR-KIs in Ph-like ALL. Disclosures Weinstock: Novartis: Consultancy, Research Funding. Mullighan:Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; Loxo Oncology: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase whose activity contributes to leukemia proliferation and survival. Compounds targeting the mTOR active site inhibit rapamycin-resistant functions and have enhanced anti-cancer activity in mouse models. MLN0128 (formerly known as INK128) is a novel, orally active mTOR kinase inhibitor currently in clinical development. Here we evaluated MLN0128 in preclinical models of B-cell acute lymphoblastic leukemia (B-ALL). MLN0128 suppressed proliferation of B-ALL cell lines in vitro and reduced colony formation by primary human leukemia cells from adult and pediatric B-ALL patients. MLN0128 also boosted the efficacy of dasatinib in Philadelphia Chromosome-positive (Ph+) specimens. In a syngeneic mouse model of lymphoid BCR-ABL+ disease, daily oral dosing of MLN0128 rapidly cleared leukemic outgrowth. In primary xenografts of Ph+ B-ALL specimens, MLN0128 significantly enhanced the efficacy of dasatinib. In non-Ph B-ALL xenografts, single agent MLN0128 had a cytostatic effect that was most pronounced in mice with low disease burden. In all in vivo models, MLN0128 was well tolerated and did not suppress endogenous bone marrow proliferation. These findings support the rationale for clinical testing of MLN0128 in both adult and pediatric B-ALL and provide insight towards optimizing therapeutic efficacy of mTOR kinase inhibitors.

Targeting the mammalian target of rapamycin (mTOR) protein is a promising strategy for cancer therapy. The mTOR kinase functions in two complexes, TORC1 (target of rapamycin complex-1) and TORC2 (target of rapamycin complex-2); however, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. We compared rapamycin with PP242, an inhibitor of the active site of mTOR in both TORC1 and TORC2 (hereafter referred to as TORC1/2), in models of acute leukemia harboring the Philadelphia chromosome (Ph) translocation. We demonstrate that PP242, but not rapamycin, causes death of mouse and human leukemia cells. In vivo, PP242 delays leukemia onset and augments the effects of the current front-line tyrosine kinase inhibitors more effectively than does rapamycin. Unexpectedly, PP242 has much weaker effects than rapamycin on the proliferation and function of normal lymphocytes. PI-103, a less selective TORC1/2 inhibitor that also targets phosphoinositide 3-kinase (PI3K), is more immunosuppressive than PP242. These findings establish that Ph(+) transformed cells are more sensitive than normal lymphocytes to selective TORC1/2 inhibitors and support the development of such inhibitors for leukemia therapy.

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.

Abstract 2585 Pre-B Acute Lymphoblastic Leukemia (B-ALL) remains a significant cause of morbidity and mortality in both adults and children. Targeting signaling pathways that sustain B-ALL cell survival is a promising strategy to improve treatment outcomes. The target of rapamycin (TOR) is a serine/threonine kinase that integrates signals from oncoproteins and the microenvironment to support ALL cell proliferation and survival. A single TOR enzyme in cells forms two multiprotein complexes, TORC1 and TORC2, with distinct regulation and substrates. The classical TOR inhibitor rapamycin inhibits only some functions of TORC1 and does not acutely inhibit TORC2. A new generation of TOR inhibitors has been developed that inhibit all outputs of both TORC1 and TORC2. These ATP-competitive, TOR kinase inhibitors (TOR-KIs) have improved anti-cancer activity in models of both solid tumors and hematological malignancies. Previously we reported that the TOR-KI compound PP242 is effective in mouse models of Philadelphia Chromosome-positive (Ph+) B-ALL, with minimal toxicity to normal hematopoietic cells or immune function (MR Janes et al., Nat. Med. 2010; 16:205). Here we present promising results on the efficacy of a second generation TOR-KI, INK128, in both Ph+ and non-Ph B-ALL. INK128 is an orally active, highly selective TOR-KI that is currently in phase I clinical trials for solid tumors, multiple myeloma and Waldenstrom's Macroglobulinemia. We find that INK128 has similar biological effects as PP242 on Ph+ B-ALL cells, but these effects are apparent at 5-fold lower concentrations in vitro and 80-fold lower doses in mice. In methylcellulose assays using Ph+ and non-Ph B-ALL specimens, INK128 greatly reduces colony numbers and enhances the efficacy of standard-of-care drugs. Both adult and pediatric non-Ph B-ALL specimens are sensitive to growth suppression by INK128. INK128 also causes apoptosis in B-ALL cells cultured on stromal cells. Importantly, INK128 suppresses human Ph+ B-ALL xenograft expansion in mice at doses that do not impair normal hematopoietic cell proliferation. INK128 is also less immunosuppressive than rapamycin. Together these results provide rationale for testing INK128 and other TOR-KIs in clinical trials of B-ALL therapy. Disclosures: Fruman: Intellikine: Membership on an entity's Board of Directors or advisory committees, Research Funding. Janes:Intellikine: Employment. Martin:Intellikine: Employment. Ren:Intellikine: Employment. Liu:Intellikine: Employment. Rommel:Intellikine: Employment.