Autophagy maintains hematopoietic stem cell integrity and prevents malignant transformation. In addition to bulk degradation, selective autophagy serves as an intracellular quality control mechanism ...and requires autophagy receptors, such as p62 (SQSTM1), to specifically bridge the ubiquitinated cargos into autophagosomes. Here, we investigated the function of p62 in acute myeloid leukemia (AML) in vitro and in murine in vivo models of AML. Loss of p62 impaired expansion and colony-forming ability of leukemia cells and prolonged latency of leukemia development in mice. High p62 expression was associated with poor prognosis in human AML. Using quantitative mass spectrometry, we identified enrichment of mitochondrial proteins upon immunoprecipitation of p62. Loss of p62 significantly delayed removal of dysfunctional mitochondria, increased mitochondrial superoxide levels, and impaired mitochondrial respiration. Moreover, we demonstrated that the autophagy-dependent function of p62 is essential for cell growth and effective mitochondrial degradation by mitophagy. Our results highlight the prominent role of selective autophagy in leukemia progression, and specifically, the importance of mitophagy to maintain mitochondrial integrity.
•Loss of the selective autophagy receptor p62 impairs leukemia development in a murine leukemia model.•p62 binds to mitochondria in leukemia cells and is required for efficient removal of damaged mitochondria by mitophagy.
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Abstract
Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs ...multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.
In many countries, a majority of cancer patients are not treated at comprehensive cancer centers. Even for those that are, parts of the treatment or follow‐up may be carried out in local community ...hospitals or in private practices. How to assure quality in cancer care and create innovation? How to integrate decentralized versus centralized patient care, education, and cancer research? Outlined here is a 360° view of outreach to include all stakeholders—most importantly patients and their families, patient advocacy groups, healthcare providers, health insurers, and policymakers.
In many countries, a majority of cancer patients are not treated at comprehensive cancer centers. Even for those that are, parts of the treatment or follow‐up may be carried out in local community hospitals or in private practices. Outlined here is a 360° view of outreach to include all stakeholders—most importantly patients and their families, patient advocacy groups, healthcare providers, health insurers, and policymakers.
The hierarchical organization of the hematopoietic system requires hematopoietic stem cells (HSCs) capable of self-renewal and multilineage differentiation to produce all cellular lineages of the ...blood. Novel techniques of purification of hematopoietic subpopulations and their functional characterization have defined individual steps of their lineage commitment. A detailed molecular map of the selective autophagy landscape governing self-renewal, maintenance, and differentiation of HSCs and their progeny during early and terminal differentiation has not yet been drawn. However, the importance of selective autophagy pathways is increasingly being recognized, as this evolutionary conserved degradation pathway is instrumental in orchestrating intracellular turnover of macromolecular complexes and organelles to meet the specific needs of various hematopoietic cells. For instance, mitophagy has clearly been demonstrated to regulate mitochondrial homeostasis in HSCs, thereby defining their metabolic fate decisions. This review focuses on the emerging evidence supporting an important role of selective autophagy in regulating normal hematopoiesis. As this has uncovered important features in malignant hematopoiesis, the review also covers aspects of selective autophagy pathways affected in the development and progression of leukemia and lymphoma. A molecular understanding of the unique requirements of selective autophagy may open new avenues for specific therapeutic intervention.
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•HSCs require autophagy, particularly mitophagy, for self-maintenance under hypoxia.•HSC fate decisions & differentiation are driven by mitochondrial metabolism.•Autophagy defects impair lineage maturation & terminal blood cell functions.•AML & other hematological malignancies require selective autophagy pathways.•Details of selective autophagy pathways in hematopoiesis are still lacking.
Purpose The initial results of the APL0406 trial showed that the combination of all- trans-retinoic acid (ATRA) and arsenic trioxide (ATO) is at least not inferior to standard ATRA and chemotherapy ...(CHT) in first-line therapy of low- or intermediate-risk acute promyelocytic leukemia (APL). We herein report the final analysis on the complete series of patients enrolled onto this trial. Patients and Methods The APL0406 study was a prospective, randomized, multicenter, open-label, phase III noninferiority trial. Eligible patients were adults between 18 and 71 years of age with newly diagnosed, low- or intermediate-risk APL (WBC at diagnosis ≤ 10 × 10
/L). Overall, 276 patients were randomly assigned to receive ATRA-ATO or ATRA-CHT between October 2007 and January 2013. Results Of 263 patients evaluable for response to induction, 127 (100%) of 127 patients and 132 (97%) of 136 patients achieved complete remission (CR) in the ATRA-ATO and ATRA-CHT arms, respectively ( P = .12). After a median follow-up of 40.6 months, the event-free survival, cumulative incidence of relapse, and overall survival at 50 months for patients in the ATRA-ATO versus ATRA-CHT arms were 97.3% v 80%, 1.9% v 13.9%, and 99.2% v 92.6%, respectively ( P < .001, P = .0013, and P = .0073, respectively). Postinduction events included two relapses and one death in CR in the ATRA-ATO arm and two instances of molecular resistance after third consolidation, 15 relapses, and five deaths in CR in the ATRA-CHT arm. Two patients in the ATRA-CHT arm developed a therapy-related myeloid neoplasm. Conclusion These results show that the advantages of ATRA-ATO over ATRA-CHT increase over time and that there is significantly greater and more sustained antileukemic efficacy of ATO-ATRA compared with ATRA-CHT in low- and intermediate-risk APL.
Balancing the processes of hematopoietic stem cell (HSC) differentiation and self-renewal is critical for maintaining a lifelong supply of blood cells. The bone marrow (BM) produces a stable output ...of newly generated cells, but immunologic stress conditions inducing leukopenia increase the demand for peripheral blood cell supply. Here we demonstrate that the proinflammatory cytokine interferon-γ (IFN-γ) impairs maintenance of HSCs by directly reducing their proliferative capacity and that IFN-γ impairs restoration of HSC numbers upon viral infection. We show that IFN-γ reduces thrombopoietin (TPO)-mediated phosphorylation of signal transducer and activator of transcription (STAT) 5, an important positive regulator of HSC self-renewal. IFN-γ also induced expression of suppressor of cytokine signaling (SOCS) 1 in HSCs, and we demonstrate that SOCS1 expression is sufficient to inhibit TPO-induced STAT5 phosphorylation. Furthermore, IFN-γ deregulates expression of STAT5-mediated cell-cycle genes cyclin D1 and p57. These findings suggest that IFN-γ is a negative modulator of HSC self-renewal by modifying cytokine responses and expression of genes involved in HSC proliferation. We postulate that the occurrence of BM failure in chronic inflammatory conditions, such as aplastic anemia, HIV, and graft-versus-host disease, is related to a sustained impairment of HSC self-renewal caused by chronic IFN-γ signaling in these disorders.
•IFN-γ impairs maintenance of HSCs by directly reducing their proliferative capacity and impairing their restoration upon viral infection.•IFN-γ induces SOCS1 expression in HSCs, which inhibits TPO-induced STAT5 phosphorylation, thereby deregulating key cell-cycle genes.
Sample loss and contamination are critical preanalytical pitfalls in microscale proteomic applications of nonadhering cells. Common assays and workflows are not easily adoptable to microscale sample ...sizes of suspension cells due to inadvertent sample loss. This impedes preanalytical experimental manipulation of limited suspension cell samples for microscale proteomics applications, such as encountered for primary human materials. Here, we describe and test a simple manual batch technique for single-step 100-fold concentration of scarce numbers of diluted suspension cells (down to 5000 cells) by volume reduction, facilitating microscale experiments with suspension cells. Pipette tips with heat-sealed orifices (SpinTips) are manufactured within 1 min and serve as versatile microcentrifugation vessels from which supernatant can be aspirated with minimal cell loss. A residual volume of approximately 3 μL can be achieved without visualization of the cell pellet. The results show that SpinTips enable the concentration, medium exchange, washing, and culture of highly limited amounts of suspension cells for functional manipulation and microscale proteomics and are readily incorporated into standard workflows. The application is illustrated by profiling ex vivo responses of primary acute myeloid leukemia (AML) cells from one AML patient to daunorubicin (DNR) to a depth of 3462 quantified proteins with excellent repeatability.
The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) ...enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials.
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The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant ...cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.
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•NPM1/NPM1c induce the autophagy-lysosome pathway by activating the master regulator TFEB•NPM1/NPM1c bind to GABARAP proteins via an atypical module in their N-terminal regions•The pro-autophagic activity of NPM1c depends on this GABARAP binding module
Mende and co-workers report that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB. They identify an atypical GABARAP binding module in the N-terminal region of NPM1 and the AML-associated oncogenic mutant NPM1c and show that the pro-autophagic activity of NPM1c depends on this module.
Abstract
Internal tandem duplications (ITD) in the receptor tyrosine kinase FLT3 occur in 25 % of acute myeloid leukemia (AML) patients, drive leukemia progression and confer a poor prognosis. ...Primary resistance to FLT3 kinase inhibitors (FLT3i) quizartinib, crenolanib and gilteritinib is a frequent clinical challenge and occurs in the absence of identifiable genetic causes. This suggests that adaptive cellular mechanisms mediate primary resistance to on-target FLT3i therapy. Here, we systematically investigated acute cellular responses to on-target therapy with multiple FLT3i in FLT3-ITD + AML using recently developed functional translatome proteomics (measuring changes in the nascent proteome) with phosphoproteomics. This pinpointed AKT-mTORC1-ULK1-dependent autophagy as a dominant resistance mechanism to on-target FLT3i therapy. FLT3i induced autophagy in a concentration- and time-dependent manner specifically in FLT3-ITD + cells in vitro and in primary human AML cells ex vivo. Pharmacological or genetic inhibition of autophagy increased the sensitivity to FLT3-targeted therapy in cell lines, patient-derived xenografts and primary AML cells ex vivo. In mice xenografted with FLT3-ITD + AML cells, co-treatment with oral FLT3 and autophagy inhibitors synergistically impaired leukemia progression and extended overall survival. Our findings identify a molecular mechanism responsible for primary FLT3i treatment resistance and demonstrate the pre-clinical efficacy of a rational combination treatment strategy targeting both FLT3 and autophagy induction.
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