The MYC-Regulated RNA-Binding Proteins hnRNPC and LARP1 Are Drivers of Multiple Myeloma Cell Growth and Disease Progression and Negatively Predict Patient Survival.

Multiple myeloma (MM) is a malignant plasma cell disorder in which the MYC oncogene is frequently dysregulated. Due to its central role, MYC has been proposed as a drug target; however, the development of a clinically applicable molecule modulating MYC activity remains an unmet challenge. Consequently, an alternative is the development of therapeutic options targeting proteins located downstream of MYC. Therefore, we aimed to identify undescribed MYC-target proteins in MM cells using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and mass spectrometry. We revealed a cluster of proteins associated with the regulation of translation initiation. Herein, the RNA-binding proteins Heterogeneous Nuclear Ribonucleoprotein C (hnRNPC) and La Ribonucleoprotein 1 (LARP1) were predominantly downregulated upon MYC depletion. CRISPR-mediated knockout of either hnRNPC or LARP1 in conjunction with redundant LARP family proteins resulted in a proliferative disadvantage for MM cells. Moreover, high expression levels of these proteins correlate with high MYC expression and with poor survival and disease progression in MM patients. In conclusion, our study provides valuable insights into MYC's role in translation initiation by identifying hnRNPC and LARP1 as proliferation drivers of MM cells and as both predictive factors for survival and disease progression in MM patients.

An atypical GABARAP binding module drives the pro-autophagic potential of the AML-associated NPM1c variant.

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.

PDP1 is a key metabolic gatekeeper and modulator of drug resistance in FLT3-ITD-positive acute myeloid leukemia.

High metabolic flexibility is pivotal for the persistence and therapy resistance of acute myeloid leukemia (AML). In 20-30% of AML patients, activating mutations of FLT3, specifically FLT3-ITD, are key therapeutic targets. Here, we investigated the influence of FLT3-ITD on AML metabolism. Nuclear Magnetic Resonance (NMR) profiling showed enhanced reshuffling of pyruvate towards the tricarboxylic acid (TCA) cycle, suggesting an increased activity of the pyruvate dehydrogenase complex (PDC). Consistently, FLT3-ITD-positive cells expressed high levels of PDP1, an activator of the PDC. Combining endogenous tagging of PDP1 with genome-wide CRISPR screens revealed that FLT3-ITD induces PDP1 expression through the RAS signaling axis. PDP1 knockdown resulted in reduced cellular respiration thereby impairing the proliferation of only FLT3-ITD cells. These cells continued to depend on PDP1, even in hypoxic conditions, and unlike FLT3-ITD-negative cells, they exhibited a rapid, PDP1-dependent revival of their respiratory capacity during reoxygenation. Moreover, we show that PDP1 modifies the response to FLT3 inhibition. Upon incubation with the FLT3 tyrosine kinase inhibitor quizartinib (AC220), PDP1 persisted or was upregulated, resulting in a further shift of glucose/pyruvate metabolism towards the TCA cycle. Overexpression of PDP1 enhanced, while PDP1 depletion diminished AC220 resistance in cell lines and peripheral blasts from an AC220-resistant AML patient in vivo. In conclusion, FLT3-ITD assures the expression of PDP1, a pivotal metabolic regulator that enhances oxidative glucose metabolism and drug resistance. Hence, PDP1 emerges as a potentially targetable vulnerability in the management of AML.

Deciphering the mitophagy receptor network identifies a crucial role for OPTN (optineurin) in acute myeloid leukemia.

The selective autophagic degradation of mitochondria via mitophagy is essential for preserving mitochondrial homeostasis and, thereby, disease maintenance and progression in acute myeloid leukemia (AML). Mitophagy is orchestrated by a variety of mitophagy receptors whose interplay is not well understood. Here, we established a pairwise multiplexed CRISPR screen targeting mitophagy receptors to elucidate redundancies and gain a deeper understanding of the functional interactome governing mitophagy in AML. We identified OPTN (optineurin) as sole non-redundant mitophagy receptor and characterized its unique role in AML. Knockdown and overexpression experiments demonstrated that OPTN expression is rate-limiting for AML cell proliferation. In a MN1-driven murine transplantation model, loss of OPTN prolonged overall median survival by 7 days (+21%). Mechanistically, we found broadly impaired mitochondrial respiration and function with increased mitochondrial ROS, that most likely caused the proliferation defect. Our results decipher the intertwined network of mitophagy receptors in AML for both ubiquitin-dependent and receptor-mediated mitophagy, identify OPTN as a non-redundant tool to study mitophagy in the context of leukemia and suggest OPTN inhibition as an attractive therapeutic strategy.Abbreviations: AML: acute myeloid leukemia; CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; CTRL: control; DFP: deferiprone; GI: genetic interaction; KD: knockdown; KO: knockout; ldMBM, lineage-depleted murine bone marrow; LFC: log2 fold change; LIR: LC3-interacting region; LSC: leukemic stem cell; MAGeCK: Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout; MDIVI-1: mitochondrial division inhibitor 1; MOI: multiplicity of infection; MOM: mitochondrial outer membrane; NAC: N-acetyl-L-cysteine; OA: oligomycin-antimycin A; OCR: oxygen consumption rate; OE: overexpression; OPTN: optineurin; PINK1: PTEN induced putative kinase 1; ROS: reactive oxygen species; SEM: standard error of the mean; TCGA: The Cancer Genome Atlas; TEM: transmission electron microscopy; UBD: ubiquitin-binding domain; WT: wild type.

Bone Marrow Assessment in Liver Cirrhosis Patients with Otherwise Unexplained Peripheral Blood Cytopenia.

We performed a retrospective single-center analysis to investigate the diagnostic yield of bone marrow puncture in patients with liver cirrhosis and cytopenia. Liver cirrhosis patients receiving bone marrow aspiration or biopsy for the diagnostic work-up of otherwise unexplained peripheral blood cytopenia at our institution between 2004 and 2020 were enrolled in this study. We evaluated findings from cytologic, histologic and immunologic assessment and final diagnostic outcomes. A total of 118 patients with a median age of 55 years and a median Child-Pugh score of B (8 points) were enrolled. The main etiologies of liver cirrhosis were viral hepatitis (B and C) or chronic alcohol consumption. The majority of patients (60%) exhibited concurrent anemia, leukocytopenia and thrombocytopenia. Bone marrow assessment revealed normal, unspecific or reactive alterations in 117 out of 118 patients (99%). One patient was diagnosed with myelodysplastic syndrome. Our findings suggest that peripheral blood cytopenia in patients with liver cirrhosis is rarely associated with a primary bone marrow pathology.

Diagnostic yield, indications, and outcomes of cranial imaging in AML patients admitted for intensive induction or consolidation chemotherapy: a single-center experience.

Cranial imaging (CI) is a widely used diagnostic procedure, especially in acute myeloid leukemia (AML) patients with suspected bleeding or infection. However, common clinical decision rules to guide CI do not apply to AML patients and the diagnostic yield and outcomes of CI for AML patients are largely unknown. We retrospectively evaluated all CI from newly diagnosed non-promyelocytic AML patients receiving intensive induction or consolidation chemotherapy between 2007 and 2019 for imaging indications, diagnostic yield, and consequences. A total of 110 of 462 patients (24%) received CI for 152 imagings in distinct clinical situations. Forty-four patients (40%) had at least one new and acute pathological finding. Main indication was focal neurologic deficit, craniocerebral trauma, and suspected cerebral hypertension. The most common new finding was intracranial hemorrhage (13% of all imagings), followed by sinusitis (9%). CI led to therapy change in 21 patients. There were no clear associations between indications, laboratory values, and a positive imaging. Positive imaging was associated with adverse overall survival. Our study suggests that the overall rate of ordered CI was appropriate and that CI should generally be performed at a low threshold. A systematized approach to CI may further increase diagnostic yield but is complicated by variable clinical presentation.

Early-onset venous thromboembolisms in newly diagnosed non-promyelocytic acute myeloid leukemia patients undergoing intensive induction chemotherapy.

OBJECTIVES AND METHODS: Venous thromboembolic (VTE) events are emerging as frequent complications in acute myeloid leukemia (AML); however, there is insufficient data regarding epidemiology, risk factors, and impact on outcomes. The optimal approach to balance risks of thrombosis and hemorrhage remains unclear. This retrospective single-center study in AML patients undergoing induction chemotherapy between 2007 and 2018 assessed incidence, risk factors, features, and outcomes of early-onset VTE. RESULTS: 423 patients (median age 59 years) were enrolled. VTE was diagnosed in 31 patients (7.3%) within 3 months of admission. The median time to VTE was 3 days. Non-central venous catheter (CVC)-related VTE occurred in 19 patients (61%). Main risk factor for VTE was leukocytosis at admission, independent of platelet counts/INR. Four patients (13%) exhibited VTE recurrence. No deaths directly related to VTE or major bleeding events associated with platelet-adjusted anticoagulation in patients with VTE were recorded. There was no clear impact of VTE on 1-year overall survival; however, non-CVC-related VTE may be associated with adverse outcomes. CONCLUSIONS: Early-onset VTE is a common complication in newly diagnosed AML patients admitted for induction chemotherapy. Leukocytosis is an independent VTE risk factor. The potentially adverse impact of non-CVC-related VTE merits further study.

SpinTip: A Simple, Robust, and Versatile Preanalytical Method for Microscale Suspension Cell Proteomics.

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.

Translatome proteomics identifies autophagy as a resistance mechanism to on-target FLT3 inhibitors in acute myeloid leukemia.

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.

Relapse surveillance of acute myeloid leukemia patients in first remission after consolidation chemotherapy: diagnostic value of regular bone marrow aspirations.

The optimal follow-up care for relapse detection in acute myeloid leukemia (AML) patients in first remission after consolidation therapy with intensive chemotherapy is not established. In this retrospective study, we evaluate the diagnostic value of an intensive relapse surveillance strategy by regular bone marrow aspirations (BMA) in these patients. We identified 86 patients with newly diagnosed non-promyelocytic AML who had reached complete remission (CR) after intensive induction and consolidation chemotherapy between 2007 and 2019. Annual relapse rates were 40%, 17%, and 2% in years 1-3, respectively. Patients in CR were surveilled by BMA scheduled every 3 months for 2 years, followed by BMA every 6 months. This surveillance regimen detected 29 of 55 relapses (53%), 11 of which were molecular relapses (20%). The remaining 26 of 55 relapses (47%) were diagnosed by non-surveillance BMA prompted by specific suspicion of relapse. Most patients showed concurrent morphological abnormalities in peripheral blood (PB) at time of relapse. Seven percent of all morphological relapses occurred without simultaneous PB abnormalities and would have been delayed without surveillance BMA. Intensified monthly PB assessment paired with BMA every 3 months during the first 2 years may be a highly sensitive relapse surveillance strategy.

Intracranial hemorrhage in newly diagnosed non-promyelocytic acute myeloid leukemia patients admitted for intensive induction chemotherapy.

OBJECTIVES AND METHODS: Intracranial hemorrhage (ICH) in acute myeloid leukemia (AML) patients is a major concern due to the increased risk of mortality. Few studies have examined ICH specifically in newly diagnosed AML patients receiving intensive induction chemotherapy (IC) and prophylactic platelet transfusions during thrombocytopenia <10/nL. This retrospective cohort study included 423 newly diagnosed AML patients without acute promyelocytic leukemia who underwent IC between 2007 and 2019. We assessed risk factors, clinical features, and outcomes of ICH. RESULTS: 17 of 423 patients (4%) suffered ICH during hospital stay, and 4 patients (24%) died directly because of ICH despite routine prophylactic platelet transfusions. Patients with ICH had a negatively impacted overall survival (median OS, 20.1 vs. 104.8 months) and were more likely not to continue with curative treatment. Main risk factors were female gender, severe thrombocytopenia, and decreased fibrinogen. Patients with subsequent ICH also had laboratory signs of liver dysfunction. CONCLUSIONS: Intracranial hemorrhage remains a potentially deadly complication with notable incidence despite prophylactic platelet substitution, suggesting that additional prophylactic interventions may be required to further reduce the frequency of ICH in high-risk patients. Unrecognized genetic factors may simultaneously predispose to AML and platelet dysfunction with ICH.

Metabolic Rewiring Is Essential for AML Cell Survival to Overcome Autophagy Inhibition by Loss of ATG3.

Autophagy is an important survival mechanism that allows recycling of nutrients and removal of damaged organelles and has been shown to contribute to the proliferation of acute myeloid leukemia (AML) cells. However, little is known about the mechanism by which autophagy- dependent AML cells can overcome dysfunctional autophagy. In our study we identified autophagy related protein 3 (ATG3) as a crucial autophagy gene for AML cell proliferation by conducting a CRISPR/Cas9 dropout screen with a library targeting around 200 autophagy-related genes. shRNA-mediated loss of ATG3 impaired autophagy function in AML cells and increased their mitochondrial activity and energy metabolism, as shown by elevated mitochondrial ROS generation and mitochondrial respiration. Using tracer-based NMR metabolomics analysis we further demonstrate that the loss of ATG3 resulted in an upregulation of glycolysis, lactate production, and oxidative phosphorylation. Additionally, loss of ATG3 strongly sensitized AML cells to the inhibition of mitochondrial metabolism. These findings highlight the metabolic vulnerabilities that AML cells acquire from autophagy inhibition and support further exploration of combination therapies targeting autophagy and mitochondrial metabolism in AML.

Fluid overload is associated with increased 90-day mortality in AML patients undergoing induction chemotherapy.

Treatment-related complications contribute substantially to morbidity and mortality in acute myeloid leukemia (AML) patients undergoing induction chemotherapy. Although AML patients are susceptible to fluid overload (FO) (e.g., in the context of chemotherapy protocols, during sepsis treatment or to prevent tumor lysis syndrome), little attention has been paid to its role in AML patients undergoing induction chemotherapy. AML patients receiving induction chemotherapy between 2014 and 2019 were included in this study. FO was defined as ≥5% weight gain on day 7 of induction chemotherapy compared to baseline weight determined on the day of admission. We found FO in 23 (12%) of 187 AML patients undergoing induction chemotherapy. Application of >100 ml crystalloid fluids/kg body weight until day 7 of induction chemotherapy was identified as an independent risk factor for FO. AML patients with FO suffered from a significantly increased 90-day mortality rate and FO was demonstrated as an independent risk factor for 90-day mortality. Our data suggests an individualized, weight-adjusted calculation of crystalloid fluids in order to prevent FO-related morbidity and mortality in AML patients during induction chemotherapy. Prospective trials are required to determine the adequate fluid management in this patient population.

Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer.

OBJECTIVES: Multidrug-resistant organisms (MDRO) are considered an emerging threat worldwide. Data covering the clinical impact of MDRO colonization in patients with solid malignancies, however, is widely missing. We sought to determine the impact of MDRO colonization in patients who have been diagnosed with Non-small cell lung cancer (NSCLC) who are at known high-risk for invasive infections. MATERIALS AND METHODS: Patients who were screened for MDRO colonization within a 90-day period after NSCLC diagnosis of all stages were included in this single-center retrospective study. RESULTS: Two hundred and ninety-five patients were included of whom 24 patients (8.1%) were screened positive for MDRO colonization (MDROpos) at first diagnosis. Enterobacterales were by far the most frequent MDRO detected with a proportion of 79.2% (19/24). MDRO colonization was present across all disease stages and more present in patients with concomitant diabetes mellitus. Median overall survival was significantly inferior in the MDROpos study group with a median OS of 7.8 months (95% CI, 0.0-19.9 months) compared to a median OS of 23.9 months (95% CI, 17.6-30.1 months) in the MDROneg group in univariate (p = 0.036) and multivariate analysis (P = 0.02). Exploratory analyses suggest a higher rate of non-cancer-related-mortality in MDROpos patients compared to MDROneg patients (p = 0.002) with an increased rate of fatal infections in MDROpos patients (p = 0.0002). CONCLUSIONS: MDRO colonization is an independent risk factor for inferior OS in patients diagnosed with NSCLC due to a higher rate of fatal infections. Empirical antibiotic treatment approaches should cover formerly detected MDR commensals in cases of (suspected) invasive infections.

Selective Autophagy in Normal and Malignant Hematopoiesis.

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.

The notch target gene HEYL modulates metastasis forming capacity of colorectal cancer patient-derived spheroid cells in vivo.

BACKGROUND: While colorectal cancer (CRC) patients with localized disease have a favorable prognosis, the five-year-survival rate in patients with distant spread is still below 15%. Hence, a detailed understanding of the mechanisms regulating metastasis formation is essential to develop therapeutic strategies targeting metastasized CRC. The notch pathway has been shown to be involved in the metastatic spread of various tumor entities; however, the impact of its target gene HEYL remains unclear so far. METHODS: In this study, we functionally assessed the association between high HEYL expression and metastasis formation in human CRC. Therefore, we lentivirally overexpressed HEYL in two human patient-derived CRC cultures differing in their spontaneous metastasizing capacity and analyzed metastasis formation as well as tumor cell dissemination into the bone marrow after xenotransplantation into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. RESULTS: HEYL overexpression decreased tumor cell dissemination and the absolute numbers of formed metastases in a sub-renal capsular spontaneous metastasis formation model, addressing all steps of the metastatic cascade. In contrast, metastatic capacity was not decreased following intrasplenic xenotransplantation where the cells are placed directly into the blood circulation. CONCLUSION: These results suggest that HEYL negatively regulates metastasis formation in vivo presumably by inhibiting intravasation of metastasis-initiating cells.

Low-dose melphalan in elderly patients with relapsed or refractory acute myeloid leukemia: A well-tolerated and effective treatment after hypomethylating-agent failure.

Relapsed or refractory (R/R) disease remains challenging in acute myeloid leukemia (AML), especially in elderly patients not considered eligible for intensive treatment options. We retrospectively evaluated the safety and efficacy of low-dose melphalan (LD-Mel) in a multicenter analysis in patients over 65 years with R/R AML, who previously had received ≥1 non-curative treatment line. The study included 31 patients (median age 77 years) with 1-4 previous treatment lines. Three patients (9.7%) achieved a complete remission. Two patients (6.5%) achieved a partial remission, nine patients (29.0%) had disease stabilization with reduction of peripheral or bone marrow blast burden, resulting in an overall response rate of 16.1% and 45.2% achieved clinical benefit. Responders showed a significantly longer median overall survival than non-responders (16.3 vs. 2.3 months, p < 0.001). Multivariate analysis identified complex karyotype as the only risk factor associated with inferior survival (p < 0.001), whereas prior treatment with hypomethylating agents (HMAs) in 25 of 31 patients was associated with superior OS, regardless of prior response to HMAs (p = 0.03). LD-Mel was well tolerated, with mild myelosuppressive side effects. Conclusively, LD-Mel is an effective treatment option in elderly patients with R/R AML, particularly after HMA therapy and in the absence of a complex karyotype.

Efficient determination of axon number in the optic nerve: A stereological approach.

Quantifying the number of axons in the optic nerve is of interest in many research questions. Here, we show that a stereological method allows simple, efficient, precise and unbiased determination of the total axon number in the murine optic nerve. Axons in semi-thin optic nerve cross sections from untreated eyes (n = 21) and eyes subjected to retinal damage by intravitreous NMDA injections (n = 32) or PBS controls (n = 5) were manually identified, counted and digitally labeled by hand. A stereological procedure was empirically tested with systematic combinations of different sampling methods (simple random sampling without replacement, systematic uniform random sampling, stratified random sampling) and sampling parameters. Extensive numerical Monte Carlo experiments were performed to evaluate their large-sample properties. Our results demonstrate reliable determination of total axon number and superior performance compared to other methods at a small fraction of the time required for a full manual count. We specify suitable sampling parameters for the adoption of an efficient stereological sampling scheme, give empirical estimates of the additionally introduced sampling variance to facilitate experimental planning, and offer AxonCounter, an easy-to-use plugin implementing these stereological methods for the multi-platform image processing application NIH ImageJ.