Amit J. Sabnis

Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects

Mutations in genes encoding ribosomal proteins cause the Minute phenotype in Drosophila and mice, and Diamond-Blackfan syndrome in humans. Here we report two mouse dark skin (Dsk) loci caused by mutations in Rps19 (ribosomal protein S19) and Rps20 (ribosomal protein S20). We identify a common pathophysiologic program in which p53 stabilization stimulates Kit ligand expression, and, consequently, epidermal melanocytosis via a paracrine mechanism. Accumulation of p53 also causes reduced body size and erythrocyte count. These results provide a mechanistic explanation for the diverse collection of phenotypes that accompany reduced dosage of genes encoding ribosomal proteins, and have implications for understanding normal human variation and human disease.

The Hippo effector YAP promotes resistance to RAF- and MEK-targeted cancer therapies

Resistance to RAF- and MEK-targeted therapy is a major clinical challenge. RAF and MEK inhibitors are initially but only transiently effective in some but not all patients with BRAF gene mutation and are largely ineffective in those with RAS gene mutation because of resistance. Through a genetic screen in BRAF-mutant tumor cells, we show that the Hippo pathway effector YAP (encoded by YAP1) acts as a parallel survival input to promote resistance to RAF and MEK inhibitor therapy. Combined YAP and RAF or MEK inhibition was synthetically lethal not only in several BRAF-mutant tumor types but also in RAS-mutant tumors. Increased YAP in tumors harboring BRAF V600E was a biomarker of worse initial response to RAF and MEK inhibition in patients, establishing the clinical relevance of our findings. Our data identify YAP as a new mechanism of resistance to RAF- and MEK-targeted therapy. The findings unveil the synthetic lethality of combined suppression of YAP and RAF or MEK as a promising strategy to enhance treatment response and patient survival.

JunB protects against myeloid malignancies by limiting hematopoietic stem cell proliferation and differentiation without affecting self-renewal.

Loss of the JunB/AP-1 transcription factor induces a myeloproliferative disease (MPD) arising from the hematopoietic stem cell (HSC) compartment. Here, we show that junB inactivation deregulates the cell-cycle machinery and increases the proliferation of long-term repopulating HSCs (LT-HSCs) without impairing their self-renewal or regenerative potential in vivo. We found that JunB loss destabilizes a complex network of genes and pathways that normally limit myeloid differentiation, leading to impaired responsiveness to both Notch and TGF-beta signaling due in part to transcriptional deregulation of the Hes1 gene. These results demonstrate that LT-HSC proliferation and differentiation are uncoupled from self-renewal and establish some of the mechanisms by which JunB normally limits the production of myeloid progenitors, hence preventing initiation of myeloid malignancies.

RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS–mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRASWT) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK–positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

Oncogenic Kras initiates leukemia in hematopoietic stem cells.

How oncogenes modulate the self-renewal properties of cancer-initiating cells is incompletely understood. Activating KRAS and NRAS mutations are among the most common oncogenic lesions detected in human cancer, and occur in myeloproliferative disorders (MPDs) and leukemias. We investigated the effects of expressing oncogenic KrasG12D from its endogenous locus on the proliferation and tumor-initiating properties of murine hematopoietic stem and progenitor cells. MPD could be initiated by KrasG12D expression in a highly restricted population enriched for hematopoietic stem cells (HSCs), but not in common myeloid progenitors. KrasG12D HSCs demonstrated a marked in vivo competitive advantage over wild-type cells. KrasG12D expression also increased the fraction of proliferating HSCs and reduced the overall size of this compartment. Transplanted KrasG12D HSCs efficiently initiated acute T-lineage leukemia/lymphoma, which was associated with secondary Notch1 mutations in thymocytes. We conclude that MPD-initiating activity is restricted to the HSC compartment in KrasG12D mice, and that distinct self-renewing populations with cooperating mutations emerge during cancer progression.

Combined chemical–genetic approach identifies cytosolic HSP70 dependence in rhabdomyosarcoma

Significance Protein chaperone networks maintain homeostasis during cellular stress. Oncogenic transformation induces stress through increased demands on protein synthesis and folding. Thus, many cancer cells depend on proteostasis networks for optimal growth. However, the cancer subtype-specific roles of individual protein chaperones are incompletely understood. Through a chemical–genetic approach, we discovered an exquisite dependence of rhabdomyosarcoma (RMS) cells on cytosolic heat-shock protein 70 kDa (HSP70). HSP70 inhibition activates the unfolded protein response, and CEBP homologous protein is a key mediator of apoptosis and a candidate biomarker for efficacy. The link between a component required for cytosolic protein quality control and the endoplasmic reticulum stress response provides insight into cell type-specific wiring of proteostasis networks and suggests novel therapeutic avenues in RMS. Cytosolic and organelle-based heat-shock protein (HSP) chaperones ensure proper folding and function of nascent and injured polypeptides to support cell growth. Under conditions of cellular stress, including oncogenic transformation, proteostasis components maintain homeostasis and prevent apoptosis. Although this cancer-relevant function has provided a rationale for therapeutically targeting proteostasis regulators (e.g., HSP90), cancer-subtype dependencies upon particular proteostasis components are relatively undefined. Here, we show that human rhabdomyosarcoma (RMS) cells, but not several other cancer cell types, depend upon heat-shock protein 70 kDA (HSP70) for survival. HSP70-targeted therapy (but not chemotherapeutic agents) promoted apoptosis in RMS cells by triggering an unfolded protein response (UPR) that induced PRKR-like endoplasmic reticulum kinase (PERK)–eukaryotic translation initiation factor α (eIF2α)–CEBP homologous protein (CHOP) signaling and CHOP-mediated cell death. Intriguingly, inhibition of only cytosolic HSP70 induced the UPR, suggesting that the essential activity of HSP70 in RMS cells lies at the endoplasmic reticulum–cytosol interface. We also found that increased CHOP mRNA in clinical specimens was a biomarker for poor outcomes in chemotherapy-treated RMS patients. The data suggest that, like human epidermal growth factor receptor 2 (HER2) amplification in breast cancer, increased CHOP in RMS is a biomarker of decreased response to chemotherapy but enhanced response to targeted therapy. Our findings identify the cytosolic HSP70–UPR axis as an unexpected regulator of RMS pathogenesis, revealing HSP70-targeted therapy as a promising strategy to engage CHOP-mediated apoptosis and improve RMS treatment. Our study highlights the utility of dissecting cancer subtype-specific dependencies on proteostasis networks to uncover unanticipated cancer vulnerabilities.

Activating Mutations Cluster in the “Molecular Brake” Regions of Protein Kinases and Do Not Associate with Conserved or Catalytic Residues

Mutations leading to activation of proto‐oncogenic protein kinases (PKs) are a type of drivers crucial for understanding tumorogenesis and as targets for antitumor drugs. However, bioinformatics tools so far developed to differentiate driver mutations, typically based on conservation considerations, systematically fail to recognize activating mutations in PKs. Here, we present the first comprehensive analysis of the 407 activating mutations described in the literature, which affect 41 PKs. Unexpectedly, we found that these mutations do not associate with conserved positions and do not directly affect ATP binding or catalytic residues. Instead, they cluster around three segments that have been demonstrated to act, in some PKs, as “molecular brakes” of the kinase activity. This finding led us to hypothesize that an auto inhibitory mechanism mediated by such “brakes” is present in all PKs and that the majority of activating mutations act by releasing it. Our results also demonstrate that activating mutations of PKs constitute a distinct group of drivers and that specific bioinformatics tools are needed to identify them in the numerous cancer sequencing projects currently underway. The clustering in three segments should represent the starting point of such tools, a hypothesis that we tested by identifying two somatic mutations in EPHA7 that might be functionally relevant.

FGFR Fusions in the Driver's Seat

Through a clinical deep sequencing protocol, Wu and colleagues have identified multiple FGFR fusion proteins in diverse cancers. Pharmacologic inhibition of FGFR suppressed the growth of FGFR fusion-positive tumor models, suggesting that these FGFR fusions are oncogenic drivers and highlighting the use of streamlined clinical sequencing efforts to identify novel, actionable driver oncoproteins in human tumors.

Reducing Second Gram-Negative Antibiotic Therapy on Pediatric Oncology and Hematopoietic Stem Cell Transplantation Services

OBJECTIVE To evaluate interventions to reduce avoidable antibiotic use on pediatric oncology and hematopoietic stem cell transplantation (HSCT) services. DESIGN Interrupted time series. SETTING Academic pediatric hospital with separate oncology and HSCT services. PARTICIPANTS Children admitted to the services during baseline (October 2011-August 2013) and 2 intervention periods, September 2013-June 2015 and July 2015-June 2016, including 1,525 oncology hospitalizations and 301 HSCT hospitalizations. INTERVENTION In phase 1, we completed an update of the institutional febrile neutropenia (FN) guideline for the pediatric oncology service, recommending first-line β-lactam monotherapy rather than routine use of 2 gram-negative agents. Phase 2 included updating the HSCT service FN guideline and engagement with a new pediatric antimicrobial stewardship program. The use of target antibiotics (tobramycin and ciprofloxacin) was measured in days of therapy per 1,000 patient days collected from administrative data. Intervention effects were evaluated using interrupted time series with segmented regression. RESULTS Phase 1 had mixed effects-long-term reduction in tobramycin use (97% below projected at 18 months) but rebound with increasing slope in ciprofloxacin use (+18% per month). Following phase 2, tobramycin and ciprofloxacin use on the oncology service were both 99% below projected levels at 12 months. On the HSCT service, tobramycin use was 99% below the projected level and ciprofloxacin use was 96% below the projected level at 12 months. CONCLUSIONS Locally adapted guidelines can facilitate practice changes in oncology and HSCT settings. More comprehensive and ongoing interventions, including follow-up education, feedback, and engagement of companion services may be needed to sustain changes. Infect Control Hosp Epidemiol 2017;38:1039-1047.

Synthetic essentiality of metabolic regulator PDHK1 in PTEN-deficient cells and cancers

PTEN is a tumor suppressor that is often inactivated in cancer and possesses both lipid and protein phosphatase activities. We report the metabolic regulator PDHK1 (pyruvate dehydrogenase kinase1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The predominant mechanism of PDHK1 regulation and dependency is the PTEN protein phosphatase dephosphorylates NFκ;B activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NFκB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to drive aerobic glycolysis and induce PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, which is a biomarker of decreased patient survival, establishing clinical relevance. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers. SIGNIFICANCE The tumor suppressor PTEN is widely inactivated in cancers and tumor syndromes. PTEN antagonizes PI3K/AKT signaling via its lipid phosphatase activity. The modest success of PI3K/AKT inhibition in PTEN-deficient cancer patients provides rationale for identifying other vulnerabilities in PTEN-deficient cancers to improve clinical outcomes. We show that PTEN-deficient cells are uniquely sensitive to PDHK1 inhibition. PTEN and PDHK1 co-suppression reduced colony formation and induced cell death in vitro and tumor regression in vivo. PDHK1 levels were high in PTEN-deficient patient tumors and associated with inferior patient survival, establishing clinical relevance. Our study identifies a PTEN-regulated signaling pathway linking the PTEN protein phosphatase to the metabolic regulator PDHK1 and provides a mechanistic basis for PDHK1 targeting in PTEN-deficient cancers.