A spatially resolved single-cell genomic atlas of the adult human breast.

The adult human breast is comprised of an intricate network of epithelial ducts and lobules that are embedded in connective and adipose tissue1-3. Although most previous studies have focused on the breast epithelial system4-6, many of the non-epithelial cell types remain understudied. Here we constructed the comprehensive Human Breast Cell Atlas (HBCA) at single-cell and spatial resolution. Our single-cell transcriptomics study profiled 714,331 cells from 126 women, and 117,346 nuclei from 20 women, identifying 12 major cell types and 58 biological cell states. These data reveal abundant perivascular, endothelial and immune cell populations, and highly diverse luminal epithelial cell states. Spatial mapping using four different technologies revealed an unexpectedly rich ecosystem of tissue-resident immune cells, as well as distinct molecular differences between ductal and lobular regions. Collectively, these data provide a reference of the adult normal breast tissue for studying mammary biology and diseases such as breast cancer.

Role for Caspase-8 in the Release of IL-1ß and Active Caspase-1 from Viable Human Monocytes during Toxoplasma gondii Infection.

Monocytes are actively recruited to sites of infection and produce the potent proinflammatory cytokine IL-1ß. We previously showed that IL-1ß release during Toxoplasma gondii infection of primary human monocytes requires the NLRP3 inflammasome and caspase-1 but is independent of gasdermin D and pyroptosis. To investigate mechanisms of IL-1ß release, we generated caspase-1, -4, -5, or -8 knockout (KO) THP-1 monocytic cells. Genetic ablation of caspase-1 or -8, but not caspase-4 or -5, decreased IL-1ß release during T. gondii infection without affecting cell death. In contrast, TNF-α and IL-6 secretion were unperturbed in caspase-8 KO cells during T. gondii infection. Dual pharmacological inhibition of caspase-8 and RIPK1 in primary monocytes also decreased IL-1ß release without affecting cell viability or parasite infection. Caspase-8 was also required for the release of active caspase-1 from T. gondii-infected cells and for IL-1ß release during infection with the related apicomplexan parasite Neospora caninum. Surprisingly, caspase-8 deficiency did not impair synthesis or cleavage of pro-IL-1ß, but resulted in the retention of mature IL-1ß within cells. Generation of gasdermin E KO and ATG7 KO THP-1 cells revealed that the release of IL-1ß was not dependent on gasdermin E or ATG7. Collectively, our data indicate that during T. gondii Infection of human monocytes, caspase-8 functions in a novel gasdermin-independent mechanism controlling IL-1ß release from viable cells. This study expands on the molecular pathways that promote IL-1ß in human immune cells and provides evidence of a role for caspase-8 in the mechanism of IL-1ß release during infection.

Targeting eIF4F translation initiation complex with SBI-756 sensitises B lymphoma cells to venetoclax.

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.

Toxoplasma gondii activates a Syk-CARD9-NF-κB signaling axis and gasdermin D-independent release of IL-1ß during infection of primary human monocytes.

IL-1ß 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-1ß; 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-1ß 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-1ß release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1ß transcripts and the production of pro-IL-1ß. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1ß production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1ß protein levels and IL-1ß release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1ß transcriptional activation. IL-1ß 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-1ß 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-1ß 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.

Statin-induced Mitochondrial Priming Sensitizes Multiple Myeloma Cells to BCL2 and MCL-1 Inhibitors.

The BCL2 inhibitor venetoclax promotes apoptosis in blood cancer cells and is approved for treatment of chronic lymphocytic leukemia and acute myeloid leukemia. However, multiple myeloma cells are frequently more dependent on MCL-1 for survival, conferring resistance to venetoclax. Here we report that mevalonate pathway inhibition with statins can overcome resistance to venetoclax in multiple myeloma cell lines and primary cells. In addition, statins sensitize to apoptosis induced by MCL-1 inhibitor, S63845. In retrospective analysis of venetoclax clinical studies in multiple myeloma, background statin use was associated with a significantly enhanced rate of stringent complete response and absence of progressive disease. Statins sensitize multiple myeloma cells to venetoclax by upregulating two proapoptotic proteins: PUMA via a p53-independent mechanism and NOXA via the integrated stress response. These findings provide rationale for prospective testing of statins with venetoclax regimens in multiple myeloma. SIGNIFICANCE: BH3 mimetics including venetoclax hold promise for treatment of multiple myeloma but rational combinations are needed to broaden efficacy. This study presents mechanistic and clinical data to support addition of pitavastatin to venetoclax regimens in myeloma. The results open a new avenue for repurposing statins in blood cancer.

Toxoplasma gondii Extends the Life Span of Infected Human Neutrophils by Inducing Cytosolic PCNA and Blocking Activation of Apoptotic Caspases.

Toxoplasma gondii is an intracellular protozoan parasite that has the remarkable ability to infect and replicate in neutrophils, immune cells with an arsenal of antimicrobial effector mechanisms. We report that T. gondii infection extends the life span of primary human peripheral blood neutrophils by delaying spontaneous apoptosis, serum starvation-induced apoptosis, and tumor necrosis alpha (TNF-α)-mediated apoptosis. T. gondii blockade of apoptosis was associated with an inhibition of processing and activation of the apoptotic caspases caspase-8 and -3, decreased phosphatidylserine exposure on the plasma membrane, and reduced cell death. We performed a global transcriptome analysis of T. gondii-infected peripheral blood neutrophils using RNA sequencing (RNA-Seq) and identified gene expression changes associated with DNA replication and DNA repair pathways, which in mature neutrophils are indicative of changes in regulators of cell survival. Consistent with the RNA-Seq data, T. gondii infection upregulated transcript and protein expression of PCNA, which is found in the cytosol of human neutrophils, where it functions as a key inhibitor of apoptotic pro-caspases. Infection of neutrophils resulted in increased interaction of PCNA with pro-caspase-3. Inhibition of this interaction with an AlkB homologue 2 PCNA-interacting motif (APIM) peptide reversed the infection-induced delay in cell death. Taken together, these findings indicate a novel strategy by which T. gondii manipulates cell life span in primary human neutrophils, which may allow the parasite to maintain an intracellular replicative niche and avoid immune clearance.IMPORTANCEToxoplasma gondii is an obligate intracellular parasite that can cause life-threatening disease in immunocompromised individuals and in the developing fetus. Interestingly, T. gondii has evolved strategies to successfully manipulate the host immune system to establish a productive infection and evade host defense mechanisms. Although it is well documented that neutrophils are mobilized during acute T. gondii infection and infiltrate the site of infection, these cells can also be actively infected by T. gondii and serve as a replicative niche for the parasite. However, there is a limited understanding of the molecular processes occurring within T. gondii-infected neutrophils. This study reveals that T. gondii extends the life span of human neutrophils by inducing the expression of PCNA, which prevents activation of apoptotic caspases, thus delaying apoptosis. This strategy may allow the parasite to preserve its replicative intracellular niche.

Reduced eIF4E function impairs B-cell leukemia without altering normal B-lymphocyte function.

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.

Targeting eIF4F translation complex sensitizes B-ALL cells to tyrosine kinase inhibition.

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.

Efficacy of a Novel Bi-Steric mTORC1 Inhibitor in Models of B-Cell Acute Lymphoblastic Leukemia.

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.

Toxoplasma gondii Dysregulates Barrier Function and Mechanotransduction Signaling in Human Endothelial Cells.

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 ß-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.

mTOR inhibition enhances efficacy of dasatinib in ABL-rearranged Ph-like B-ALL.

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 enhance efficacy of venetoclax in blood cancers.

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.

The Selective Phosphoinoside-3-Kinase p110δ Inhibitor IPI-3063 Potently Suppresses B Cell Survival, Proliferation, and Differentiation.

The class I phosphoinoside-3-kinases (PI3Ks) are important enzymes that relay signals from cell surface receptors to downstream mediators driving cellular functions. Elevated PI3K signaling is found in B cell malignancies and lymphocytes of patients with autoimmune disease. The p110δ catalytic isoform of PI3K is a rational target since it is critical for B lymphocyte development, survival, activation, and differentiation. In addition, activating mutations in PIK3CD encoding p110δ cause a human immunodeficiency known as activated PI3K delta syndrome. Currently, idelalisib is the only selective p110δ inhibitor that has been FDA approved to treat certain B cell malignancies. p110δ inhibitors can suppress autoantibody production in mouse models, but limited clinical trials in human autoimmunity have been performed with PI3K inhibitors to date. Thus, there is a need for additional tools to understand the effect of pharmacological inhibition of PI3K isoforms in lymphocytes. In this study, we tested the effects of a potent and selective p110δ inhibitor, IPI-3063, in assays of B cell function. We found that IPI-3063 potently reduced mouse B cell proliferation, survival, and plasmablast differentiation while increasing antibody class switching to IgG1, almost to the same degree as a pan-PI3K inhibitor. Similarly, IPI-3063 potently inhibited human B cell proliferation in vitro. The p110γ isoform has partially overlapping roles with p110δ in B cell development, but little is known about its role in B cell function. We found that the p110γ inhibitor AS-252424 had no significant impact on B cell responses. A novel dual p110δ/γ inhibitor, IPI-443, had comparable effects to p110δ inhibition alone. These findings show that p110δ is the dominant isoform mediating B cell responses and establish that IPI-3063 is a highly potent molecule useful for studying p110δ function in immune cells.

mTORC1 Inhibition Induces Resistance to Methotrexate and 6-Mercaptopurine in Ph+ and Ph-like 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.

The 4E-BP-eIF4E axis promotes rapamycin-sensitive growth and proliferation in lymphocytes.

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.

mTOR kinase inhibitors synergize with histone deacetylase inhibitors to kill B-cell acute lymphoblastic leukemia cells.

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.

Resistance to mTOR kinase inhibitors in lymphoma cells lacking 4EBP1.

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.

Effects of novel isoform-selective phosphoinositide 3-kinase inhibitors on natural killer cell function.

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.

Effective and selective targeting of leukemia cells using a TORC1/2 kinase inhibitor.

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.