The essential chaperone DNAJC17 activates HSP70 to coordinate RNA splicing and G2-M progression.

Molecular chaperones including the heat-shock protein 70-kilodalton (HSP70) family and the J-domain containing protein (JDP) co-chaperones maintain homeostatic balance in eukaryotic cells through regulation of the proteome. The expansive JDP family helps direct specific HSP70 functions, and yet loss of single JDP-encoding genes is widely tolerated by mammalian cells, suggesting a high degree of redundancy. By contrast, essential JDPs might carry out HSP70-independent functions or fill cell-context dependent, highly specialized roles within the proteostasis network. Using a genetic screen of JDPs in human cancer cell lines, we found the RNA recognition motif (RRM) containing DNAJC17 to be pan-essential and investigated the contribution of its structural domains to biochemical and cellular function. We found that the RRM exerts an auto-inhibitory effect on the ability of DNAJC17 to allosterically activate ATP hydrolysis by HSP70. The J-domain, but neither the RRM nor a distal C-terminal alpha helix are required to rescue cell viability after loss of endogenous DNAJC17 . Knockdown of DNAJC17 leads to relatively few conserved changes in the abundance of individual mRNAs, but instead deranges gene expression through exon skipping, primarily of genes involved in cell cycle progression. Concordant with cell viability experiments, the C-terminal portions of DNAJC17 are dispensable for restoring splicing and G2-M progression. Overall, our findings identify essential cellular JDPs and suggest that diversification in JDP structure extends the HSP70-JDP system to control divergent processes such as RNA splicing. Future investigations into the structural basis for auto-inhibition of the DNAJC17 J-domain and the molecular regulation of splicing by these components may provide insights on how conserved biochemical mechanisms can be programmed to fill unique, non-redundant cellular roles and broaden the scope of the proteostasis network.

Clinical Targeted Next-Generation Panel Sequencing Reveals MYC Amplification Is a Poor Prognostic Factor in Osteosarcoma.

PURPOSE: Osteosarcoma risk stratification, on the basis of the presence of metastatic disease at diagnosis and histologic response to chemotherapy, has remained unchanged for four decades, does not include genomic features, and has not facilitated treatment advances. We report on the genomic features of advanced osteosarcoma and provide evidence that genomic alterations can be used for risk stratification. MATERIALS AND METHODS: In a primary analytic patient cohort, 113 tumor and 69 normal samples from 92 patients with high-grade osteosarcoma were sequenced with OncoPanel, a targeted next-generation sequencing assay. In this primary cohort, we assessed the genomic landscape of advanced disease and evaluated the correlation between recurrent genomic events and outcome. We assessed whether prognostic associations identified in the primary cohort were maintained in a validation cohort of 86 patients with localized osteosarcoma tested with MSK-IMPACT. RESULTS: In the primary cohort, 3-year overall survival (OS) was 65%. Metastatic disease, present in 33% of patients at diagnosis, was associated with poor OS (P = .04). The most frequently altered genes in the primary cohort were TP53, RB1, MYC, CCNE1, CCND3, CDKN2A/B, and ATRX. Mutational signature 3 was present in 28% of samples. MYC amplification was associated with a worse 3-year OS in both the primary cohort (P = .015) and the validation cohort (P = .012). CONCLUSION: The most frequently occurring genomic events in advanced osteosarcoma were similar to those described in prior reports. MYC amplification, detected with clinical targeted next-generation sequencing panel tests, is associated with poorer outcomes in two independent cohorts.

Rare FGFR Oncogenic Alterations in Sequenced Pediatric Solid and Brain Tumors Suggest FGFR Is a Relevant Molecular Target in Childhood Cancer.

PURPOSE: Multiple FGFR inhibitors are currently in clinical trials enrolling adults with different solid tumors, while very few enroll pediatric patients. We determined the types and frequency of FGFR alterations (FGFR1-4) in pediatric cancers to inform future clinical trial design. METHODS: Tumors with FGFR alterations were identified from two large cohorts of pediatric solid tumors subjected to targeted DNA sequencing: The Dana-Farber/Boston Children's Profile Study (n = 888) and the multi-institution GAIN/iCAT2 (Genomic Assessment Improves Novel Therapy) Study (n = 571). Data from the combined patient population of 1,395 cases (64 patients were enrolled in both studies) were reviewed and cases in which an FGFR alteration was identified by OncoPanel sequencing were further assessed. RESULTS: We identified 41 patients with tumors harboring an oncogenic FGFR alteration. Median age at diagnosis was 8 years (range, 6 months-26 years). Diagnoses included 11 rhabdomyosarcomas, nine low-grade gliomas, and 17 other tumor types. Alterations included gain-of-function sequence variants (n = 19), amplifications (n = 10), oncogenic fusions (FGFR3::TACC3 [n = 3], FGFR1::TACC1 [n = 1], FGFR1::EBF2 [n = 1], FGFR1::CLIP2 [n = 1], and FGFR2::CTNNA3 [n = 1]), pathogenic-leaning variants of uncertain significance (n = 4), and amplification in combination with a pathogenic-leaning variant of uncertain significance (n = 1). Two novel FGFR1 fusions in two different patients were identified in this cohort, one of whom showed a response to an FGFR inhibitor. CONCLUSION: In summary, activating FGFR alterations were found in approximately 3% (41/1,395) of pediatric solid tumors, identifying a population of children with cancer who may be eligible and good candidates for trials evaluating FGFR-targeted therapy. Importantly, the genomic and clinical data from this study can help inform drug development in accordance with the Research to Accelerate Cures and Equity for Children Act.

GATOR2-dependent mTORC1 activity is a therapeutic vulnerability in FOXO1 fusion-positive rhabdomyosarcoma.

Oncogenic FOXO1 gene fusions drive a subset of rhabdomyosarcoma (RMS) with poor survival; to date, these cancer drivers are therapeutically intractable. To identify new therapies for this disease, we undertook an isogenic CRISPR-interference screen to define PAX3-FOXO1-specific genetic dependencies and identified genes in the GATOR2 complex. GATOR2 loss in RMS abrogated aa-induced lysosomal localization of mTORC1 and consequent downstream signaling, slowing G1-S cell cycle transition. In vivo suppression of GATOR2 impaired the growth of tumor xenografts and favored the outgrowth of cells lacking PAX3-FOXO1. Loss of a subset of GATOR2 members can be compensated by direct genetic activation of mTORC1. RAS mutations are also sufficient to decouple mTORC1 activation from GATOR2, and indeed, fusion-negative RMS harboring such mutations exhibit aa-independent mTORC1 activity. A bisteric, mTORC1-selective small molecule induced tumor regressions in fusion-positive patient-derived tumor xenografts. These findings highlight a vulnerability in FOXO1 fusion-positive RMS and provide rationale for the clinical evaluation of bisteric mTORC1 inhibitors, currently in phase I testing, to treat this disease. Isogenic genetic screens can, thus, identify potentially exploitable vulnerabilities in fusion-driven pediatric cancers that otherwise remain mostly undruggable.

Molecular profiling identifies targeted therapy opportunities in pediatric solid cancer.

To evaluate the clinical impact of molecular tumor profiling (MTP) with targeted sequencing panel tests, pediatric patients with extracranial solid tumors were enrolled in a prospective observational cohort study at 12 institutions. In the 345-patient analytical population, median age at diagnosis was 12 years (range 0-27.5); 298 patients (86%) had 1 or more alterations with potential for impact on care. Genomic alterations with diagnostic, prognostic or therapeutic significance were present in 61, 16 and 65% of patients, respectively. After return of the results, impact on care included 17 patients with a clarified diagnostic classification and 240 patients with an MTP result that could be used to select molecularly targeted therapy matched to identified alterations (MTT). Of the 29 patients who received MTT, 24% had an objective response or experienced durable clinical benefit; all but 1 of these patients received targeted therapy matched to a gene fusion. Of the diagnostic variants identified in 209 patients, 77% were gene fusions. MTP with targeted panel tests that includes fusion detection has a substantial clinical impact for young patients with solid tumors.

Entrectinib in children and young adults with solid or primary CNS tumors harboring NTRK, ROS1, or ALK aberrations (STARTRK-NG).

BACKGROUND: Entrectinib is a TRKA/B/C, ROS1, ALK tyrosine kinase inhibitor approved for the treatment of adults and children aged ≥12 years with NTRK fusion-positive solid tumors and adults with ROS1 fusion-positive non-small-cell lung cancer. We report an analysis of the STARTRK-NG trial, investigating the recommended phase 2 dose (RP2D) and activity of entrectinib in pediatric patients with solid tumors including primary central nervous system tumors. METHODS: STARTRK-NG (NCT02650401) is a phase 1/2 trial. Phase 1, dose-escalation of oral, once-daily entrectinib, enrolled patients aged <22 years with solid tumors with/without target NTRK1/2/3, ROS1, or ALK fusions. Phase 2, basket trial at the RP2D, enrolled patients with intracranial or extracranial solid tumors harboring target fusions or neuroblastoma. Primary endpoints: phase 1, RP2D based on toxicity; phase 2, objective response rate (ORR) in patients harboring target fusions. Safety-evaluable patients: ≥1 dose of entrectinib; response-evaluable patients: measurable/evaluable baseline disease and ≥1 dose at RP2D. RESULTS: At data cutoff, 43 patients, median age of 7 years, were response-evaluable. In phase 1, 4 patients experienced dose-limiting toxicities. The most common treatment-related adverse event was weight gain (48.8%). Nine patients experienced bone fractures (20.9%). In patients with fusion-positive tumors, ORR was 57.7% (95% CI 36.9-76.7), median duration of response was not reached, and median (interquartile range) duration of treatment was 10.6 months (4.2-18.4). CONCLUSIONS: Entrectinib resulted in rapid and durable responses in pediatric patients with solid tumors harboring NTRK1/2/3 or ROS1 fusions.

Clinical characteristics and outcomes of infants compared with children diagnosed with rhabdomyosarcoma: Analysis of surveillance, epidemiology and end results data from 2000 to 2016.

BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, but occurs infrequently in infants (<1 year). Historically, infants with RMS have worse overall survival compared to other pediatric age groups. AIM: This study aims to assess the clinical features and treatment factors associated with survival comparing infants to children aged 1-9 years diagnosed with RMS. METHODS: Children aged <10 years diagnosed with RMS between 2000 and 2016 were identified using the SEER database. Descriptive statistics were used to assess demographic, clinical, and treatment characteristics of infants and children with RMS. Kaplan-Meier estimates and Cox proportional hazards regression were performed to assess for factors associated with survival. RESULTS: Age <1 year was independently associated with an increased risk of mortality. Compared to children aged 1-9 years, fewer infants received standard of care therapy, that is, chemotherapy combined with local control (surgery and/or radiation; 86.8 vs. 75.7%; p = .009). In comparing the frequency of specific treatment modalities (used alone or in combination with other modalities), infants were less likely to receive radiation therapy (34.0 vs. 66.4%; p < .001) and more likely to receive surgery (68.9 vs. 57.5%; p = .02) than children aged 1-9 years. Across age groups, chemotherapy combined with local control was significantly associated with reduced mortality. Alveolar histology, metastatic disease, and Hispanic ethnicity were negatively associated with survival. CONCLUSIONS: Age of <1 year was an independent risk factor for increased mortality from RMS compared to ages 1-9 years. Fewer infants were treated with chemotherapy combined with local control, the therapy associated with best survival in all age groups. Other factors contributing to differences in survival should be further explored.

Germline Sequencing Improves Tumor-Only Sequencing Interpretation in a Precision Genomic Study of Patients With Pediatric Solid Tumor.

PURPOSE: Molecular tumor profiling is becoming a routine part of clinical cancer care, typically involving tumor-only panel testing without matched germline. We hypothesized that integrated germline sequencing could improve clinical interpretation and enhance the identification of germline variants with significant hereditary risks. MATERIALS AND METHODS: Tumors from pediatric patients with high-risk, extracranial solid malignancies were sequenced with a targeted panel of cancer-associated genes. Later, germline DNA was analyzed for a subset of these genes. We performed a post hoc analysis to identify how an integrated analysis of tumor and germline data would improve clinical interpretation. RESULTS: One hundred sixty participants with both tumor-only and germline sequencing reports were eligible for this analysis. Germline sequencing identified 38 pathogenic or likely pathogenic variants among 35 (22%) patients. Twenty-five (66%) of these were included in the tumor sequencing report. The remaining germline pathogenic or likely pathogenic variants were single-nucleotide variants filtered out of tumor-only analysis because of population frequency or copy-number variation masked by additional copy-number changes in the tumor. In tumor-only sequencing, 308 of 434 (71%) single-nucleotide variants reported were present in the germline, including 31% with suggested clinical utility. Finally, we provide further evidence that the variant allele fraction from tumor-only sequencing is insufficient to differentiate somatic from germline events. CONCLUSION: A paired approach to analyzing tumor and germline sequencing data would be expected to improve the efficiency and accuracy of distinguishing somatic mutations and germline variants, thereby facilitating the process of variant curation and therapeutic interpretation for somatic reports, as well as the identification of variants associated with germline cancer predisposition.

Use of three-dimensional printing and intraoperative navigation in the surgical resection of metastatic acetabular osteosarcoma.

A 21-year-old man underwent a joint-preserving posterior acetabular resection of metastatic osteosarcoma using a three-dimensional (3D) printed model and intraoperative navigation. The combined application of these advanced technologies can allow for surgical planning of osteotomies involving complex anatomy and help guide resections intraoperatively. They can maximise the achievement of negative oncological margins, preservation of native hip stability and critical neurovascular structures, and optimal postoperative function in an effort to resect all clinically evident disease. For this particular patient, with secondary bony metastases, they allowed for a safe and well-tolerated procedure that ultimately afforded him palliative benefit, improved quality of life and, conceivably, prolonged survival in the setting of a devastating prognosis. Although he, sadly, has since passed away, he survived for over 2 years after initial metastasis with preserved hip stability and the ability to graduate college, stay active and maintain a quality of life that addressed his goals of care.

Synthetic Essentiality of Metabolic Regulator PDHK1 in PTEN-Deficient Cells and Cancers.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor and bi-functional lipid and protein phosphatase. We report that the metabolic regulator pyruvate dehydrogenase kinase1 (PDHK1) is a synthetic-essential gene in PTEN-deficient cancer and normal cells. The PTEN protein phosphatase dephosphorylates nuclear factor κB (NF-κB)-activating protein (NKAP) and limits NFκB activation to suppress expression of PDHK1, a NF-κB target gene. Loss of the PTEN protein phosphatase upregulates PDHK1 to induce aerobic glycolysis and PDHK1 cellular dependence. PTEN-deficient human tumors harbor increased PDHK1, a biomarker of decreased patient survival. This study uncovers a PTEN-regulated signaling pathway and reveals PDHK1 as a potential target in PTEN-deficient cancers.

Principles of Resistance to Targeted Cancer Therapy: Lessons from Basic and Translational Cancer Biology.

Identification of the genomic drivers of cancer has led to the clinical development of targeted therapies that strike at the heart of many malignancies. Nonetheless, many cancers outsmart such precision-medicine efforts, and thus therapeutic resistance contributes significantly to cancer mortality. Attempts to understand the basis for resistance in patient samples and laboratory models has yielded two major benefits: one, more effective chemical inhibitors and rational combination therapies are now employed to prevent or circumvent resistance pathways; and two, our understanding of how oncogenic mutations drive cancer cell survival and oncogene addiction is deeper and broader, highlighting downstream or parallel cellular programs that shape these phenotypes. This review discusses emerging principles of resistance to therapies targeted against key oncogenic drivers.

Compensatory increases of select proteostasis networks after Hsp70 inhibition in cancer cells.

Cancer cells thrive when challenged with proteotoxic stress by inducing components of the protein folding, proteasome, autophagy and unfolded protein response (UPR) pathways. Consequently, specific molecular chaperones have been validated as targets for anti-cancer therapies. For example, inhibition of Hsp70 family proteins (hereafter Hsp70) in rhabdomyosarcoma triggers UPR induction and apoptosis. To define how these cancer cells respond to compromised proteostasis, we compared rhabdomyosarcoma cells that were sensitive (RMS13) or resistant (RMS13-R) to the Hsp70 inhibitor MAL3-101. We discovered that endoplasmic reticulum-associated degradation (ERAD) and autophagy were activated in RMS13-R cells, suggesting that resistant cells overcome Hsp70 ablation by increasing misfolded protein degradation. Indeed, RMS13-R cells degraded ERAD substrates more rapidly than RMS cells and induced the autophagy pathway. Surprisingly, inhibition of the proteasome or ERAD had no effect on RMS13-R cell survival, but silencing of select autophagy components or treatment with autophagy inhibitors restored MAL3-101 sensitivity and led to apoptosis. These data indicate a route through which cancer cells overcome a chaperone-based therapy, define how cells can adapt to Hsp70 inhibition, and demonstrate the value of combined chaperone and autophagy-based therapies.This article has an associated First Person interview with the first author of the paper.

EWSR1-NFATC2 gene fusion in a soft tissue tumor with epithelioid round cell morphology and abundant stroma: a case report and review of the literature.

Mesenchymal round cell tumors are a diverse group of neoplasms defined by primitive, often high-grade cytomorphology. The most common molecular alterations detected in these tumors are gene rearrangements involving EWSR1 to one of many fusion partners. Rare EWSR1-NFATC2 gene rearrangements, corresponding to a t(20;22) gene translocation, have been described in mesenchymal tumors with clear round cell morphology and a predilection for the skeleton. We present a case of a tumor harboring the EWSR1-NFATC2 gene fusion arising in the subcutaneous tissue of a young woman. The tumor exhibited corded and trabecular architecture of epithelioid cells within abundant myxoid and fibrous stroma. The cells showed strong immunoreactivity for NKX2.2, variable CD99, keratin, and epithelial membrane antigen, but were negative for S100 and myoepithelial markers. Importantly, similar to previously reported cases, the clinical course was more indolent than that of Ewing sarcoma. This case highlights the distinctive clinicopathological characteristics of EWSR1-NFATC2 gene fusion-associated neoplasms that distinguish them from Ewing sarcoma.

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 ß-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.

Combined chemical-genetic approach identifies cytosolic HSP70 dependence in rhabdomyosarcoma.

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.

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.

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.

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.