The picky mTORC1 in metabolic enzyme degradation.

In this issue of Molecular Cell, Yi et al.1 demonstrate that reduced mTORC1 activity induces the CTLH E3 ligase-dependent degradation of HMGCS1, an enzyme in the mevalonate pathway, thus revealing a unique connection between mTORC1 signaling and the degradation of a specific metabolic enzyme via the ubiquitin-proteasome system.

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.

PI3K/AKT/mTOR signaling transduction pathway and targeted therapies in cancer.

The PI3K/AKT/mTOR (PAM) signaling pathway is a highly conserved signal transduction network in eukaryotic cells that promotes cell survival, cell growth, and cell cycle progression. Growth factor signalling to transcription factors in the PAM axis is highly regulated by multiple cross-interactions with several other signaling pathways, and dysregulation of signal transduction can predispose to cancer development. The PAM axis is the most frequently activated signaling pathway in human cancer and is often implicated in resistance to anticancer therapies. Dysfunction of components of this pathway such as hyperactivity of PI3K, loss of function of PTEN, and gain-of-function of AKT, are notorious drivers of treatment resistance and disease progression in cancer. In this review we highlight the major dysregulations in the PAM signaling pathway in cancer, and discuss the results of PI3K, AKT and mTOR inhibitors as monotherapy and in co-administation with other antineoplastic agents in clinical trials as a strategy for overcoming treatment resistance. Finally, the major mechanisms of resistance to PAM signaling targeted therapies, including PAM signaling in immunology and immunotherapies are also discussed.

The road less traveled: activating an oncogenic kinase.

Elevated phosphoinositide 3-kinase (PI3K) activity in human tumors has prompted widespread efforts to develop chemical PI3K inhibitors for oncology indications. In an innovative new study, Gong et al. report the discovery of a highly selective activator of the PI3Kα isoform, with promising activity in assays of nerve regrowth and cardioprotection from ischemia-reperfusion injury (IRI).

Novel pharmacological and dietary approaches to target mTOR in B-cell acute lymphoblastic leukemia.

High-risk subtypes of B-cell acute lymphoblastic leukemia (B-ALL) are frequently associated with aberrant activation of tyrosine kinases (TKs). These include Ph+ B-ALL driven by BCR-ABL, and Ph-like B-ALL that carries other chromosomal rearrangements and/or gene mutations that activate TK signaling. Currently, the tyrosine kinase inhibitor (TKI) dasatinib is added to chemotherapy as standard of care in Ph+ B-ALL, and TKIs are being tested in clinical trials for Ph-like B-ALL. However, growth factors and nutrients in the leukemia microenvironment can support cell cycle and survival even in cells treated with TKIs targeting the driving oncogene. These stimuli converge on the kinase mTOR, whose elevated activity is associated with poor prognosis. In preclinical models of Ph+ and Ph-like B-ALL, mTOR inhibitors strongly enhance the anti-leukemic efficacy of TKIs. Despite this strong conceptual basis for targeting mTOR in B-ALL, the first two generations of mTOR inhibitors tested clinically (rapalogs and mTOR kinase inhibitors) have not demonstrated a clear therapeutic window. The aim of this review is to introduce new therapeutic strategies to the management of Ph-like B-ALL. We discuss novel approaches to targeting mTOR in B-ALL with potential to overcome the limitations of previous mTOR inhibitor classes. One approach is to apply third-generation bi-steric inhibitors that are selective for mTOR complex-1 (mTORC1) and show preclinical efficacy with intermittent dosing. A distinct, non-pharmacological approach is to use nutrient restriction to target signaling and metabolic dependencies in malignant B-ALL cells. These two new approaches could potentiate TKI efficacy in Ph-like leukemia and improve survival.

Synthesis of a Complex Brasilicardin Analogue Utilizing a Cobalt-Catalyzed MHAT-Induced Radical Bicyclization Reaction.

We designed and executed an expedient synthesis of a complex analogue of the potent immunosuppressive natural product brasilicardin A. Our successful synthesis featured application of our recently developed MHAT-initiated radical bicyclization, which delivered the targeted, complex analogue in 17 steps in the longest linear sequence. Unfortunately, this analogue showed no observable immunosuppressive activity, which speaks to the importance of the structural and stereochemical elements of the natural core scaffold.

Simultaneous inhibition of endocytic recycling and lysosomal fusion sensitizes cells and tissues to oligonucleotide therapeutics.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

Nanocomplexes of doxorubicin and DNA fragments for efficient and safe cancer chemotherapy.

Cancer-targeted therapy by a chemotherapeutic agent formulated in a nanoscale platform has been challenged by complex and inefficient manufacturing, low drug loading, difficult characterization, and marginally improved therapeutic efficacy. This study investigated facile-to-produce nanocomplexes of doxorubicin (DOX), a widely used cancer drug, and clinically approved DNA fragments that are extracted from a natural source. DOX was found to self-assemble DNA fragments into relatively monodispersed nanocomplexes with a diameter of ∼70 nm at 14.3% (w/w) drug loading by simple and scalable mixing. The resulting DOX/DNA nanocomplexes showed sustained DOX release, unlike overly stable Doxil®, cellular uptake via multiple endocytosis pathways, and high hematological and immunological compatibility. DOX/DNA nanocomplexes eradicated EL4 T lymphoma cells in a time-dependent manner, eventually surpassing free DOX. Extended circulation of DOX/DNA nanocomplexes, while avoiding off-target accumulation in the lung and being cleared from the liver, resulted in rapid accumulation in tumor and lowered cardio toxicity. Finally, tumor growth of EL4-challenged C57BL/6 mice (syngeneic model) and OPM2-challenged NSG mice (human xenograft model) were efficiently inhibited by DOX/DNA nanocomplexes with enhanced overall survival, in comparison with free DOX and Doxil®, especially upon repeated administrations. DOX/DNA nanocomplexes are a promising chemotherapeutics delivery platform for their ease of manufacturing, high biocompatibility, desired drug release and accumulation, efficient tumor eradication with improved safety, and further engineering versatility for extended therapeutic applications.

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.

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.

A cross-institutional analysis of the effects of broadening trainee professional development on research productivity.

PhD-trained scientists are essential contributors to the workforce in diverse employment sectors that include academia, industry, government, and nonprofit organizations. Hence, best practices for training the future biomedical workforce are of national concern. Complementing coursework and laboratory research training, many institutions now offer professional training that enables career exploration and develops a broad set of skills critical to various career paths. The National Institutes of Health (NIH) funded academic institutions to design innovative programming to enable this professional development through a mechanism known as Broadening Experiences in Scientific Training (BEST). Programming at the NIH BEST awardee institutions included career panels, skill-building workshops, job search workshops, site visits, and internships. Because doctoral training is lengthy and requires focused attention on dissertation research, an initial concern was that students participating in additional complementary training activities might exhibit an increased time to degree or diminished research productivity. Metrics were analyzed from 10 NIH BEST awardee institutions to address this concern, using time to degree and publication records as measures of efficiency and productivity. Comparing doctoral students who participated to those who did not, results revealed that across these diverse academic institutions, there were no differences in time to degree or manuscript output. Our findings support the policy that doctoral students should participate in career and professional development opportunities that are intended to prepare them for a variety of diverse and important careers in the workforce.

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.

Targeting the Mevalonate Pathway in Cancer.

The mevalonate synthesis inhibitors, statins, are mainstay therapeutics for cholesterol management and cardiovascular health. Thirty years of research have uncovered supportive roles for the mevalonate pathway in numerous cellular processes that support oncogenesis, most recently macropinocytosis. Central to the diverse mechanisms of statin sensitivity is an acquired dependence on one mevalonate pathway output, protein geranylgeranylation. New chemical prenylation probes and the discovery of a novel geranylgeranyl transferase hold promise to deepen our understanding of statin mechanisms of action. Further, insights into statin selection and the counterproductive role of dietary geranylgeraniol highlight how we should assess statins in the clinic. Lastly, rational combination strategies preview how statins will enter the oncology toolbox.

A Case for Phosphoinositide 3-Kinase-Targeted Therapy for Infectious Disease.

PI3Ks activate critical signaling cascades and have multifaceted regulatory functions in the immune system. Loss-of-function and gain-of-function mutations in the PI3Kδ isoform have revealed that this enzyme can substantially impact immune responses to infectious agents and their products. Moreover, reports garnered from decades of infectious disease studies indicate that pharmacologic inhibition of the PI3K pathway could potentially be effective in limiting the growth of certain microbes via modulation of the immune system. In this review, we briefly highlight the development and applications of PI3K inhibitors and summarize data supporting the concept that PI3Kδ inhibitors initially developed for oncology have immune regulatory potential that could be exploited to improve the control of some infectious diseases. This repurposing of existing kinase inhibitors could lay the foundation for alternative infectious disease therapy using available therapeutic agents.

YAP-mediated mechanotransduction tunes the macrophage inflammatory response.

Macrophages are innate immune cells that adhere to the extracellular matrix within tissues. However, how matrix properties regulate their function remains poorly understood. Here, we report that the adhesive microenvironment tunes the macrophage inflammatory response through the transcriptional coactivator YAP. We find that adhesion to soft hydrogels reduces inflammation when compared to adhesion on stiff materials and is associated with reduced YAP expression and nuclear localization. Substrate stiffness and cytoskeletal polymerization, but not adhesive confinement nor contractility, regulate YAP localization. Furthermore, depletion of YAP inhibits macrophage inflammation, whereas overexpression of active YAP increases inflammation. Last, we show in vivo that soft materials reduce expression of inflammatory markers and YAP in surrounding macrophages when compared to stiff materials. Together, our studies identify YAP as a key molecule for controlling inflammation and sensing stiffness in macrophages and may have broad implications in the regulation of macrophages in health and disease.

Dietary glutamine supplementation suppresses epigenetically-activated oncogenic pathways to inhibit melanoma tumour growth.

Tumour cells adapt to nutrient deprivation in vivo, yet strategies targeting the nutrient poor microenvironment remain unexplored. In melanoma, tumour cells often experience low glutamine levels, which promote cell dedifferentiation. Here, we show that dietary glutamine supplementation significantly inhibits melanoma tumour growth, prolongs survival in a transgenic melanoma mouse model, and increases sensitivity to a BRAF inhibitor. Metabolomic analysis reveals that dietary uptake of glutamine effectively increases the concentration of glutamine in tumours and its downstream metabolite, αKG, without increasing biosynthetic intermediates necessary for cell proliferation. Mechanistically, we find that glutamine supplementation uniformly alters the transcriptome in tumours. Our data further demonstrate that increase in intra-tumoural αKG concentration drives hypomethylation of H3K4me3, thereby suppressing epigenetically-activated oncogenic pathways in melanoma. Therefore, our findings provide evidence that glutamine supplementation can serve as a potential dietary intervention to block melanoma tumour growth and sensitize tumours to targeted therapy via epigenetic reprogramming.

An integrative model of pathway convergence in genetically heterogeneous blast crisis chronic myeloid leukemia.

Targeted therapies against the BCR-ABL1 kinase have revolutionized treatment of chronic phase (CP) chronic myeloid leukemia (CML). In contrast, management of blast crisis (BC) CML remains challenging because BC cells acquire complex molecular alterations that confer stemness features to progenitor populations and resistance to BCR-ABL1 tyrosine kinase inhibitors. Comprehensive models of BC transformation have proved elusive because of the rarity and genetic heterogeneity of BC, but are important for developing biomarkers predicting BC progression and effective therapies. To better understand BC, we performed an integrated multiomics analysis of 74 CP and BC samples using whole-genome and exome sequencing, transcriptome and methylome profiling, and chromatin immunoprecipitation followed by high-throughput sequencing. Employing pathway-based analysis, we found the BC genome was significantly enriched for mutations affecting components of the polycomb repressive complex (PRC) pathway. While transcriptomically, BC progenitors were enriched and depleted for PRC1- and PRC2-related gene sets respectively. By integrating our data sets, we determined that BC progenitors undergo PRC-driven epigenetic reprogramming toward a convergent transcriptomic state. Specifically, PRC2 directs BC DNA hypermethylation, which in turn silences key genes involved in myeloid differentiation and tumor suppressor function via so-called epigenetic switching, whereas PRC1 represses an overlapping and distinct set of genes, including novel BC tumor suppressors. On the basis of these observations, we developed an integrated model of BC that facilitated the identification of combinatorial therapies capable of reversing BC reprogramming (decitabine+PRC1 inhibitors), novel PRC-silenced tumor suppressor genes (NR4A2), and gene expression signatures predictive of disease progression and drug resistance in CP.

The mTORC1/4E-BP/eIF4E Axis Promotes Antibody Class Switching in B Lymphocytes.

During an adaptive immune response, activated mature B cells give rise to Ab-secreting plasma cells to fight infection. B cells undergo Ab class switching to produce different classes of Abs with varying effector functions. The mammalian/mechanistic target of rapamycin (mTOR) signaling pathway is activated during this process, and disrupting mTOR complex 1 (mTORC1) in B cells impairs class switching by a poorly understood mechanism. In particular, it is unclear which mTORC1 downstream substrates control this process. In this study, we used an in vitro murine model in which the mTORC1 inhibitor rapamycin, when added after a B cell has committed to divide, suppresses class switching while preserving proliferation. Investigation of mTORC1 substrates revealed a role for eukaryotic translation initiation factor 4E (eIF4E) and eIF4E-binding proteins in class switching. Mechanistically, we show that genetic or pharmacological disruption of eIF4E binding to eIF4G reduced cap-dependent translation, which specifically affected the expression of activation-induced cytidine deaminase protein but not Aicda mRNA. This translational impairment decreased Ab class switching independently of proliferation. These results uncover a previously undescribed role for mTORC1 and the eIF4E-binding proteins/eIF4E axis in activation-induced cytidine deaminase protein expression and Ab class switching in mouse B cells, suggesting that cap-dependent translation regulates key steps in B cell differentiation.