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Sara De Santis,1 Cecilia Monaldi,1 Manuela Mancini,2 Samantha Bruno,1 Michele Cavo,1,2 Simona Soverini1 1Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale, UO Ematologia ‘Lorenzo e Ariosto Seràgnoli’, Università di Bologna, Bologna, Italy; 2IRCCS Azienda Ospedaliero-Universitaria di Bologna, Istituto di Ematologia “Seràgnoli”, Bologna, ItalyCorrespondence: Sara De SantisInsitute of Hematology “Lorenzo e Ariosto Seràgnoli”, Via Massarenti 9, Bologna, 40138, ItalyTel +39 051 2143791Fax +39 051 2144037Email sara.desantis9@unibo.itAbstract: Protein kinases (PKs) play crucial roles in cellular proliferation and survival, hence their deregulation is a common event in the pathogenesis of solid and hematologic malignancies. Targeting PKs has been a promising strategy in cancer treatment, and there are now a variety of approved anticancer drugs targeting PKs. However, the phenomenon of resistance remains an obstacle to be addressed and overcoming resistance is a goal to be achieved. Chronic myeloid leukemia (CML) is the first as well as one of the best examples of a cancer that can be targeted by molecular therapy; hence, it can be used as a model disease for other cancers. This review aims to summarize up-to-date knowledge on the main mechanisms implicated in resistance to PK inhibitory therapies and to outline the main strategies that are being explored to overcome resistance. The importance of molecular diagnostics and disease monitoring in counteracting resistance will also be discussed.Keywords: protein kinases, chronic myeloid leukemia, tyrosine kinase inhibitors, synthetic lethality

This paper examines a youth oral history project conducted by/with/for immigrant youth of color and educators. Designed as a longitudinal five year project of critical participatory action research and youth oral histories, we sought initially to document generational experiences of schooling inequity, aggressive policing, housing precarity and immigration struggles. As a research collective we then confronted and chose to interrogate how COVID19, uprisings and activism, and remote learning affect youth of color. In our analysis we “discovered” the power of culturally responsive and sustaining education as a framework to cultivate critical consciousness and civic engagement. With an epistemic commitment to “no research on us without us,” decolonizing methodologies and research for social action, we review in this article our theoretical frameworks, epistemic commitments, methodologies, our ethical praxis and our evidence-based activism, as we explore the intimate details of critical participation as core to anti-racist developmental science.

Background Myelofibrosis (MF) is a clonal disorder of hemopoietic stem/progenitor cells (HSPCs) with high prevalence in elderly patients and mutations in three driver genes (JAK2, MPL, or CALR). Around 10–15% of patients are triple-negative (TN) for the three driver mutations and display significantly worse survival. Circulating extracellular vesicles (EVs) play a role in intercellular signaling and are increased in inflammation and cancer. To identify a biomolecular signature of TN patients, we comparatively evaluated the circulating HSPCs and their functional interplay with the microenvironment focusing on EV analysis. Methods Peripheral blood was collected from MF patients (n = 29; JAK2V617F mutation, n = 23; TN, n = 6) and healthy donors (HD, n = 10). Immunomagnetically isolated CD34+ cells were characterized by gene expression profiling analysis (GEP), survival, migration, and clonogenic ability. EVs were purified from platelet-poor plasma by ultracentrifugation, quantified using the Nanosight technology and phenotypically characterized by flow cytometry together with microRNA expression. Migration and survival of CD34+ cells from patients were also analyzed after in vitro treatments with selected inflammatory factors, i.e. (Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α, IL6) or after co-culture with EVs from MF patients/HD. Results The absolute numbers of circulating CD34+ cells were massively increased in TN patients. We found that TN CD34+ cells show in vitro defective functions and are unresponsive to the inflammatory microenvironment. Of note, the plasma levels of crucial inflammatory cytokines are mostly within the normal range in TN patients. Compared to JAK2V617F-mutated patients, the GEP of TN CD34+ cells revealed distinct signatures in key pathways such as survival, cell adhesion, and inflammation. Importantly, we observed the presence of mitochondrial components within plasma EVs and a distinct phenotype in TN-derived EVs compared to the JAK2V617F-mutated MF patients and HD counterparts. Notably, TN EVs promoted the survival of TN CD34+ cells. Along with a specific microRNA signature, the circulating EVs from TN patients are enriched with miR-361-5p. Conclusions Distinct EV-driven signals from the microenvironment are capable to promote the TN malignant hemopoiesis and their further investigation paves the way toward novel therapeutic approaches for rare MF.

One of the indications for BCR::ABL1 mutation testing in chronic myeloid leukemia (CML) is when tyrosine kinase inhibitor therapy (TKI) needs to be changed for unsatisfactory response. In this study, we evaluated a droplet digital PCR (ddPCR)-based multiplex strategy for the detection and quantitation of transcripts harbouring mutations conferring resistance to second-generation TKIs (2GTKIs). Parallel quantitation of e13a2, e14a2 and e1a2 BCR::ABL1 fusion transcripts enables to express results as percentage of mutation positive- over total BCR::ABL1 transcripts. We determined the limit of blank in 60 mutation-negative samples. Accuracy was demonstrated by further analysis of 48 samples already studied by next generation sequencing (NGS). Mutations could be called down to 0.5% and across 3-logs of BCR::ABL1 levels. Retrospective review of BCR::ABL1 NGS results in 513 consecutive CML patients with non-optimal response to first- or second-line TKI therapy suggested that a ddPCR-based approach targeted against 2GTKI-resistant mutations would score samples as mutation-negative in 22% of patients with warning response to imatinib but only in 6% of patients with warning response to 2GTKIs. We conclude ddPCR represents an attractive method for easy, accurate and rapid screening for 2GTKI-resistant mutations impacting on TKI selection, although ddPCR cannot identify compound mutations.

Systemic mastocytosis (SM) is due to the pathologic accumulation of neoplastic mast cells in one or more extracutaneous organ(s). Although midostaurin, a multikinase inhibitor active against both wild-type and D816V-mutated KIT, improves organ damage and symptoms, a proportion of patients relapse or have resistant disease. It is well known that Aurora kinase A (AKA) over-expression promotes tumorigenesis, but its role in the pathogenesis of systemic mastocytosis (SM) has not yet been investigated. Evidence from the literature suggests that AKA may confer cancer cell chemo-resistance, inhibit p53, and enhance Polo-like kinase 1 (Plk1), CDK1, and cyclin B1 to promote cell cycle progression. In this study, we aimed to investigate the pathogenetic role of AKA and Plk1 in the advanced forms of SM. We demonstrate here, for the first time, that SM cell lines display hyper-phosphorylated AKA and Plk1. Danusertib (Aurora kinase inhibitor) and volasertib (Plk1 inhibitor) inhibited growth and induced apoptotic cell death in HMC-1.1 and -1.2 cells. Their growth-inhibitory effects were associated with cell cycle arrest and the activation of apoptosis. Cell cycle arrest was associated with increased levels of phospho-Wee1. Wee1 inhibition by MK1775 after 24 h treatment with danusertib or volasertib, when cells were arrested in G2 phase and Wee1, was overexpressed and hyper-activated, resulting in a significantly higher rate of apoptosis than that obtained from concomitant treatment with danusertib or volasertib + MK1775 for 48 h. In conclusion, Plk1 and AKA, alone or together with Wee1, are attractive therapeutic targets in neoplastic MCs. Repurposing Plk1 or AKA ± Wee1 inhibitors in advanced clinical development for other indications is a therapeutic strategy worthy of being explored, in order to improve the outcome of patients with advanced SM.

BackgroundIn chronic myeloid leukemia (CML), Aurora kinase A and Polo like kinase 1 (Plk1), two serine-threonine kinases involved in the maintenance of a functional G2/M checkpoint, may cooperate with the constitutive tyrosine-kinase (TK) activity of the BCR-ABL1 fusion protein increasing DNA damage, promoting the occurrence of additional genomic alterations ultimately driving resistance to TK inhibitors (TKIs) and progression from chronic phase to blast crisis (BC). MethodsIn this study, we propose a new therapeutic strategy based on the combination of Aurora kinase A or Plk1 inhibition with danusertib or volasertib, respectively, and WEE1 inhibition with AZD1775. Apoptotic cell death was quantified by annexin V/propidium iodide staining and flow cytometry. Cell cycle distribution was assessed by PI staining and flow cytometry. Protein expression and activation was assessed by Western Blotting (WB) and Immunofluorescence. Clonogenic assays were performed to evaluate cells sensitivity to the different drugs.ResultsDanusertib and volasertib used as single drugs induced apoptosis and G2/M-phase arrest, associated with accumulation of phospho-WEE1. Subsequent addition of the WEE1 inhibitor AZD1775 in combination significantly enhanced the induction of apoptotic cell death in TKI-sensitive and -resistant cell lines as compared to both danusertib and volasertib alone and to the simultaneous combination. This schedule indeed induced a significant increase of the DNA double-strand break marker γH2AX, forcing the cells through successive replication cycles ultimately resulting in apoptosis. Finally, combination of danusertib or volasertib+AZD1775 significantly reduced the clonogenic potential of CD34+ CML progenitors. ConclusionsOur results may have implications for the development of innovative therapeutic approaches based on Aurora kinase A or Plk1 inhibition associated with WEE1 blocking, aimed to improve the outcomes of patients with multi-TKI-resistant or BC CML.

Over the past decade we have witnessed major advances in the molecular characterization of systemic mastocytosis (SM). This has provided important information for a better understanding of the pathogenesis of the disease but has also practically impacted on the way we diagnose and manage it. Advances in molecular testing has indeed run in parallel with advances in therapeutic targeting of constitutive active KIT, the major driver of the disease. Assessing the molecular landscape in each individual SM patient is nowadays important for diagnosis, prognosis, treatment and monitoring of therapeutic efficacy. This is facilitated by the routine availability of novel technologies like digital PCR and NGS. This review aims to summarize the pathogenesis of the disease, discuss the value of molecular diagnostic testing and how it should be performed, and provide an overview of present and future therapeutic concepts based on fine molecular characterization of SM patients.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.

Chronic myeloid leukemia (CML) is a classical example of stem cell cancer since it arises in a multipotent hematopoietic stem cell upon the acquisition of the t(9;22) chromosomal translocation, that converts it into a leukemic stem cell (LSC). The resulting BCR-ABL1 fusion gene encodes a deregulated tyrosine kinase that is recognized as the disease driver. Therapy with tyrosine kinase inhibitors (TKIs) eliminates progenitor and more differentiated cells but fails to eradicate quiescent LSCs. Thus, although many patients obtain excellent responses and a proportion of them can even attempt treatment discontinuation (treatment free remission [TFR]) after some years of therapy, LSCs persist, and represent a potentially dangerous reservoir feeding relapse and hampering TFR. Over the past two decades, intensive efforts have been devoted to the characterization of CML LSCs and to the dissection of the cell-intrinsic and -extrinsic mechanisms sustaining their persistence, in an attempt to find druggable targets enabling LSC eradication. Here we provide an overview and an update on these mechanisms, focusing in particular on the most recent acquisitions. Moreover, we provide a critical appraisal of the clinical relevance and feasibility of LSC targeting in CML.

We synthesized five novel tryptamine derivatives characterized by the presence of an azelayl chain or of a 1,1,1-trichloroethyl group, in turn connected to another heterocyclic scaffold. The combination of tryptamin-, 1,1,1-trichloroethyl- and 2-aminopyrimidinyl- moieties produced compound 9 identified as the most active compound in hematological cancer cell lines (IC50 = 0.57&ndash, 65.32 &mu, M). Moreover, keeping constant the presence of the tryptaminic scaffold and binding it to the azelayl moiety, the compounds maintain biological activity. Compound 13 is still active against hematological cancer cell lines and shows a selective effect only on HT29 cells (IC50 = 0.006 &micro, M) among solid tumor models. Compound 14 loses activity on all leukemic lines, while showing a high level of toxicity on all solid tumor lines tested (IC50 0.0015&ndash, 0.469 &micro, M).

Context: Genetic/genomic instability is a hallmark of CML. SETD2, a histone methyltransferase that trimethylates histone H3 on K36 (H3K36me3), has recently demonstrated a crucial role in preserving genomic integrity by modulating DNA Mismatch Repair (MMR) and Homologous Recombination (HR) repair. By Western blotting (WB), we previously observed SETD2 and H3K36me3 loss (resulting from aberrant SETD2 turnover) in 85% of blast crisis (BC) CML patients but not in newly diagnosed chronic phase (CP) patients who will achieve optimal responses. Objectives: 1. To investigate whether SETD2 is involved in the maintenance of genetic/genomic stability in CML. 2. To assess whether SETD2 loss precedes or follows progression from CP to blast crisis BC. Materials: SETD2-proficient (LAMA84) and -deficient (KCL22) CML cell lines and primary patient samples (n=86) were studied. Results: To investigate whether SETD2/H3K36me3 loss impinges on the activation and proficiency of HR, we used UV rays to induce DNA damage in SETD2 siRNA-depleted LAMA 84 (SETD2-proficient) cells. Compared to control cells, cells silenced for SETD2 displayed a marked increase in γH2AX (a marker of DNA damage) and fewer RAD51 foci (markers of ongoing HR). Assessment of MMR proficiency is ongoing. To further confirm the role of SETD2 as a tumor suppressor implicated in maintaining genomic stability in CML, we transfected KCL22 (SETD2-deficient) cells with an ectopic SETD2 plasmid. Preliminary results showed that SETD2 re-expression induced a reduction in cell doubling time, an accumulation of cells at G1/S checkpoint and a significant reduction in clonogenic potential. Studies are ongoing to assess the effects on DNA damage repair pathways. We next wondered when during disease history SETD2/H3K36me3 loss occurs. WB analysis ofpaired (diagnosis/progression) samples from four CML patients suggested that patients who will progress still have intact and functional SETD2 at diagnosis. Additional analyses are ongoing to assess whether SETD2/H3K36me3 loss precedes, hence allows to predict, progression to BC. Conclusion: Loss of SETD2/H3K36me3 is a novel, BCR-ABL1-independent mechanism of genetic instability in CML. Further investigations are needed to establish to what extent SETD2/H3K36me3 loss contributes to (and may be predictive of) disease progression. Supported by AIRC project 23001.

Context: Constitutively active KIT tyrosine kinase harboring the D816V mutation is the major driver of systemic mastocytosis (SM) pathogenesis. We have previously reported that the loss of function of SETD2 histone methyltransferase due to reduced protein stability is a common cooperating event in advanced SM (advSM). Objective: To dissect the mechanisms of action of midostaurin in advSM and to explore the potential of SETD2 as a marker of response to therapy. Materials: The mast cell (MC) leukemia cell line HMC-1 (negative for SETD2) and primary neoplastic MCs from 10 advSM patients, collected at regular time points during midostaurin treatment. Results: This study started from our preliminary observations indicating that aurora kinase A (AKA) is overexpressed and hyperactivated in advSM and is implicated in SETD2 proteasomal degradation. The new gold standard of therapy in advSM is midostaurin, which targets KIT andseveral other kinases, including AKA. We, thus, investigated whether midostaurin treatment may result in efficient AKA inhibition and subsequent SETD2 rescue. To this purpose, the HMC-1 cell line was treated with 5 µM midostaurin for 24 h, and phospho-AKA (T288), SETD2, and H3K36me3 (surrogate marker of SETD2 activity) were evaluated by WB. Midostaurin reduced AKA phosphorylation by 60%, partially restoring SETD2 expression and activity. We next wondered whether the same happens in patients treated with midostaurin. We, thus, collected and performed WB analysis on neoplastic mast cells from advSM patients before and during midostaurin treatment. We found that midostaurin may target AKA and rescue SETD2 expression and function in vivo and that SETD2/H3K36me3 rescue correlates with the clinical response to midostaurin and the reduction of KIT D816V allele burden, as assessed by digital PCR at 3, 6, and 12 months. In 8 patients who responded to midostaurin, SETD2/H3K36me3 rescue was detected by WB as early as 3 months after treatment onset, whereas 2 refractory patients did not show any increase in SETD2/H3K36me3 and maintained an unaltered KIT allele burden. Conclusions: Midostaurin treatment induces AKA partial dephosphorylation and consequent rescue of SETD2 expression and function in vitro and in vivo SETD2/H3K36me3 may serve as a biomarker of response to midostaurin. Supported by AIRC project 23001

The world is experiencing an unprecedented global pandemic of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus, Severe Acute Respiratory Syndrome-coronavirus-2 (SARS-CoV-2). Development of new vaccines and therapeutics are important to achieve long-term prevention and control of the virus. Experience gained in the development of vaccines for Ebola virus disease provide important lessons in the regulatory, clinical, and manufacturing process that can be applied to SARS-CoV-2 and other epidemic pathogens. This report outlines the main lessons learned by Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA (MSD) during development of an Ebola Zaire vaccine (ERVEBO®) and looks ahead to critical lessons beyond vaccine development. It highlights focus areas for public-private partnership and regulatory harmonization that can be directly applied to current vaccine development efforts for SARS-CoV-2, while drawing attention to the need for parallel consideration of issues beyond development that are equally important to achieve global preparedness and response goals.

Tumor protein p53 is a key regulator of several cellular pathways, including DNA repair, cell cycle and angiogenesis. Kevetrin exhibits p53-dependent as well as-independent activity in solid tumors, while its effects on leukemic cells remain unknown. The aim of the present study was to analyze the response of acute myeloid leukemia (AML) cell lines (TP53 wild-type: OCI-AML3 and MOLM-13; and TP53-mutant: KASUMI-1 and NOMO-1) to kevetrin at a concentration range of 85–340 µM. The cellular and molecular effects of the treatment were analyzed in terms of cell growth, viability [Annexin V-propidium iodide (PI) staining] and cell cycle alterations (PI staining). Gene expression profiling, western blotting and immunofluorescence were performed to elucidate the pathways underlying kevetrin activity. Pulsed exposure exerted no effect on the wild-type cells, but was effective on mutant cells. After continuous treatment, significant cell growth arrest and apoptosis were observed in all cell lines, with TP53-mutant models displaying a higher sensitivity and p53 induction. Kevetrin also displayed efficacy against TP53 wild-type and mutant primary AML, with a preferential cytotoxic activity against blast cells. Gene expression profiling revealed a common core transcriptional program altered by drug exposure and the downregulation of glycolysis, DNA repair and unfolded protein response signatures. These findings suggest that kevetrin may be a promising therapeutic option for patients with both wild-type and TP53-mutant AML.

In recent years, molecular characterization and management of patients with systemic mastocytosis (SM) have greatly benefited from the application of advanced technologies. Highly sensitive and accurate assays for KIT D816V mutation detection and quantification have allowed the switch to non-invasive peripheral blood testing for patient screening; allele burden has prognostic implications and may be used to monitor therapeutic efficacy. Progress in genetic profiling of KIT, together with the use of next-generation sequencing panels for the characterization of associated gene mutations, have allowed the stratification of patients into three subgroups differing in terms of pathogenesis and prognosis: i) patients with mast cell-restricted KIT D816V; ii) patients with multilineage KIT D816V-involvement; iii) patients with “multi-mutated disease”. Thanks to these findings, new prognostic scoring systems combining clinical and molecular data have been developed. Finally, non-genetic SETD2 histone methyltransferase loss of function has recently been identified in advanced SM. Assessment of SETD2 protein levels and activity might provide prognostic information and has opened new research avenues exploring alternative targeted therapeutic strategies. This review discusses how progress in recent years has rapidly complemented previous knowledge improving the molecular characterization of SM, and how this has the potential to impact on patient diagnosis and management.

Aneuploidy is a very rare and tissue-specific event in normal conditions, occurring in a low number of brain and liver cells. Its frequency increases in age-related disorders and is one of the hallmarks of cancer. Aneuploidy has been associated with defects in the spindle assembly checkpoint (SAC). However, the relationship between chromosome number alterations, SAC genes and tumor susceptibility remains unclear. Here, we provide a comprehensive review of SAC gene alterations at genomic and transcriptional level across human cancers and discuss the oncogenic and tumor suppressor functions of aneuploidy. SAC genes are rarely mutated but frequently overexpressed, with a negative prognostic impact on different tumor types. Both increased and decreased SAC gene expression show oncogenic potential in mice. SAC gene upregulation may drive aneuploidization and tumorigenesis through mitotic delay, coupled with additional oncogenic functions outside mitosis. The genomic background and environmental conditions influence the fate of aneuploid cells. Aneuploidy reduces cellular fitness. It induces growth and contact inhibition, mitotic and proteotoxic stress, cell senescence and production of reactive oxygen species. However, aneuploidy confers an evolutionary flexibility by favoring genome and chromosome instability (CIN), cellular adaptation, stem cell-like properties and immune escape. These properties represent the driving force of aneuploid cancers, especially under conditions of stress and pharmacological pressure, and are currently under investigation as potential therapeutic targets. Indeed, promising results have been obtained from synthetic lethal combinations exploiting CIN, mitotic defects, and aneuploidy-tolerating mechanisms as cancer vulnerability.

Background. Extracellular vesicles (EVs) are submicron vesicles released from various cell types including blood cells with pleiotropic effects on cell signalling and metabolism. EV cargos are enriched in nucleic acids, proteins, and lipids that can be delivered to target cells to influence surrounding microenvironment. Thus, EVs represent a powerful tool for liquid biopsy in hematological malignancies including acute myeloid leukemia (AML). AML is an aggressive disease with high relapse rate and less invasive tools are urgently needed to investigate disease (metabolic) dynamics. Accumulating evidence has reported a key role for EVs in shaping the AML bone marrow niche. However, at present, the metabolic function and the lipidomic signature driven by circulating EVs have yet to fully emerge. Methods. Peripheral blood (PB) and bone marrow (BM) were collected from AML patients at diagnosis (n=40) and PB from age/sex-matched healthy donors (HD, n=20). EVs were purified from platelet-poor plasma by size exclusion chromatography and quantified using the NanoSight technology. Immunomagnetically isolated CD34+ cells from umbilical cord blood (CB) or AML patients were characterized by analyzing the hematopoietic stem/progenitor cell (HSPC)-specific cluster of differentiation marker expression, redox metabolic profiling (using CellROX, glutathione detection reagent and MitoTracker) after 24 hours co-culture with EVs. Quantitative lipidomic profiling of circulating EVs was performed by Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC/HRMS). Seahorse extracellular flux analyses were performed in leukemia cell lines (including KG-1, KASUMI-1, MOLM-13, THP-1 and OCI-AML3). To functionally define the metabolic reprogramming of leukemic cellular components within their microenvironment, leukemic stem cell subsets were assessed by flow cytometry-based SCENITH (Single Cell ENergetic metabolism by profilIng Translation inHibition) method in both whole blood and BM samples (n=4). Results. In our work, plasma-derived EVs from AML patients showed a significant increase in the size and protein amounts compared to HD counterparts. To explore the metabolic perturbation triggered by EVs, we developed a co-culture system with circulating EVs from either HD or AML patients with CB or AML CD34+. We found a reduction in the frequency of AML CD34+ with high ROS levels in the presence of AML EVs without affecting the ROS levels in normal CB CD34+. In parallel, AML EVs increased the frequency of AML CD34+ with both high mitochondrial activity and glutathione, a key antioxidant molecule involved in many metabolic pathways. Similar metabolic profiles were also confirmed in human leukemic cell lines tested. Specifically, Seahorse flux analysis revealed that EVs induced a cell energy phenotype consistent with quiescent and chemoresistant state in human leukemic cell lines, showing a more glycolytic state in MOLM-13. Interestingly, both CD34+ and CD34+/CD38- leukemic fractions from whole blood and BM of the same AML patients were analyzed by SCENITH after co-cultures with HD/AML EVs. Remarkably, PB CD34+/CD38- leukemic fractions were more dependent on mitochondrial activity in the presence of AML EVs, suggesting a metabolic shift triggered by leukemic EV that apparently occur in the leukemic fractions out of the BM niche. In addition, to give insights into lipidomic signatures of EVs as disease biomarkers, we detected a total of 25 (out of 200) independent lipid species significantly different between AML-derived EVs and HD (n=20, respectively). We reported the abundance of both glycerolipid and fatty acids species in AML EVs. Also, through a multivariate statistical analysis of EV lipidomic profile, we revealed that AML EVs were depleted in sphingomyelin classes, a class of lipids that are interconnected to HSC metabolism. Finally, according to the 2017 ELN risk stratification system, we observed the depletion in important modulators of EV release and formation as ether-linked phosphatidylethanolamine and phosphatidylethanolamine species in adverse-risk AML patients. Conclusion. Overall, our study provides the basis for further investigations on the metabolic alterations trigger by EVs within the BM microenvironment and suggests prognostic biomarkers for leukemic patients that might reveal novel metabolic vulnerabilities in AML scenario. Disclosures Cavo: Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Curti: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees.

Context: The hypoxic bone marrow microenvironment plays a crucial role in acute myeloid leukemia (AML) development and progression, acting as a sanctuary for leukemic stem cells (LSCs). Hypoxia drives transcriptomic and metabolomic changes that enhance the fitness of LSCs, reducing their susceptibility to drug treatment. Aurora kinases A and B are crucial actors during mitosis and their overexpression has been described in AML, making them promising therapeutic targets. Phase II studies of Aurora kinase inhibitors combined with induction chemotherapy have demonstrated good tolerability and efficacy in patients with high-risk AML. Objective: We aimed to investigate the influence of hypoxia on AML sensitivity to Aurora kinase inhibition. Materials: AML cell lines (NOMO-1: t(9;11), p53 mutated; MOLM-13: FLT3-ITD; MV-4-11: t(4;11), +8, +19, FLT3-ITD; KASUMI-1 t(8;21), c-KIT and p53 mutated) were treated under either normoxia or hypoxia (1% O2) with the pan-Aurora inhibitor tozasertib (100 and 250 nM for 24 and 48 hours). Annexin V and propidium iodide (PI) staining were performed to evaluate the induction of apoptosis and proliferation capacity. Protein level alterations were analyzed by Western blotting (WB). Results: Hypoxia strongly influenced the anti-leukemic effects of tozasertib, reducing its ability to inhibit growth and proliferation in AML cells. In particular, all tested AML cell lines cultured under hypoxia showed reduced sensitivity to tozasertib compared to those under normoxia, with a significant reduction of apoptotic death after 48h of treatment. AnnexinV+ cells ranged between 32 and 60% under hypoxia vs 47-70% under normoxia (p Conclusion: Our study shows that, irrespective of the genetic background, cellular responses to Aurora inhibition in AML are markedly impaired by hypoxia. This suggests that AML LSCs might not be eradicated by tozasertib treatment.

Introduction New faculty orientation (NFO) for onboarding faculty members is replete with critical information and resources. Most NFO sessions are facilitated through lecture-based slide presentations. We employed a case-based learning (CBL) format to promote learner engagement through active use of the content being taught. We hypothesized that a CBL format would lead to improved learner satisfaction. Methods An inventory of topics presented at NFO was generated by reviewing previous orientation sessions. Cases and discussion questions were developed to link to specific content areas from previously delivered lectures. Participants were assigned cases with discussion questions, followed by a large-group debriefing. In 2014, 105 faculty members participated in NFO. An additional 232 faculty members participated from 2014 to 2017 with the revised format. Results Participants completed evaluations following each session. A specific item gauged the effectiveness of the format on a 9-point Likert scale. Scores were compared to 2014 evaluations using the unpaired Student t test. In 2014, participants rated the program format as 7.17 (mean) out of 9. Faculty orientation sessions in 2015, 2016, and 2017 scored 7.90 (p < .05), 8.00 (p < .05), and 8.40 (p < .01), respectively. Discussion We proposed a new format for NFO using a CBL-based format. Feedback highlighted faculty satisfaction with the new format. Qualitative feedback suggested that this new design in NFO sessions promotes learner engagement, networking, and collegiality. The format also models a pedagogy that faculty can implement directly in their respective teaching environments.

Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. Methods: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. Results: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919, p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. Conclusion: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.

Background Aneuploidy occurs in more than 20% of acute myeloid leukemia (AML) cases and correlates with an adverse prognosis. Methods To understand the molecular bases of aneuploid acute myeloid leukemia (A‐AML), this study examined the genomic profile in 42 A‐AML cases and 35 euploid acute myeloid leukemia (E‐AML) cases. Results A‐AML was characterized by increased genomic complexity based on exonic variants (an average of 26 somatic mutations per sample vs 15 for E‐AML). The integration of exome, copy number, and gene expression data revealed alterations in genes involved in DNA repair (eg, SLX4IP, RINT1, HINT1, and ATR) and the cell cycle (eg, MCM2, MCM4, MCM5, MCM7, MCM8, MCM10, UBE2C, USP37, CK2, CK3, CK4, BUB1B, NUSAP1, and E2F) in A‐AML, which was associated with a 3‐gene signature defined by PLK1 and CDC20 upregulation and RAD50 downregulation and with structural or functional silencing of the p53 transcriptional program. Moreover, A‐AML was enriched for alterations in the protein ubiquitination and degradation pathway (eg, increased levels of UHRF1 and UBE2C and decreased UBA3 expression), response to reactive oxygen species, energy metabolism, and biosynthetic processes, which may help in facing the unbalanced protein load. E‐AML was associated with BCOR/BCORL1 mutations and HOX gene overexpression. Conclusions These findings indicate that aneuploidy‐related and leukemia‐specific alterations cooperate to tolerate an abnormal chromosome number in AML, and they point to the mitotic and protein degradation machineries as potential therapeutic targets., Aneuploid acute myeloid leukemia (A‐AML) is associated with genomic and transcriptional alterations in the cell cycle and protein degradation pathways. The upregulation of PLK1 and CDC20 and the downregulation of RAD50 and of a p53‐related signature are hallmarks of A‐AML.

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795∗; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.

Approximately 18% of acute myeloid leukemia (AML) cases express a fusion transcript. However, few fusions are recurrent across AML and the identification of these rare chimeras is of interest to characterize AML patients. Here, we studied the transcriptome of 8 adult AML patients with poorly described chromosomal translocation(s), with the aim of identifying novel and rare fusion transcripts. We integrated RNA-sequencing data with multiple approaches including computational analysis, Sanger sequencing, fluorescence in situ hybridization and in vitro studies to assess the oncogenic potential of the ZEB2-BCL11B chimera. We detected 7 different fusions with partner genes involving transcription factors (OAZ-MAFK, ZEB2-BCL11B), tumor suppressors (SAV1-GYPB, PUF60-TYW1, CNOT2-WT1) and rearrangements associated with the loss of NF1 (CPD-PXT1, UTP6-CRLF3). Notably, ZEB2-BCL11B rearrangements co-occurred with FLT3 mutations and were associated with a poorly differentiated or mixed phenotype leukemia. Although the fusion alone did not transform murine c-Kit+ bone marrow cells, 45.4% of 14q32 non-rearranged AML cases were also BCL11B-positive, suggesting a more general and complex mechanism of leukemogenesis associated with BCL11B expression. Overall, by combining different approaches, we described rare fusion events contributing to the complexity of AML and we linked the expression of some chimeras to genomic alterations hitting known genes in AML.

Acute myeloid leukemia (AML) is a hematologic malignancy caused by a wide range of alterations responsible for a high grade of heterogeneity among patients. Several studies have demonstrated that the hypoxic bone marrow microenvironment (BMM) plays a crucial role in AML pathogenesis and therapy response. This review article summarizes the current literature regarding the effects of the dynamic crosstalk between leukemic stem cells (LSCs) and hypoxic BMM. The interaction between LSCs and hypoxic BMM regulates fundamental cell fate decisions, including survival, self-renewal, and proliferation capacity as a consequence of genetic, transcriptional, and metabolic adaptation of LSCs mediated by hypoxia-inducible factors (HIFs). HIF-1α and some of their targets have been associated with poor prognosis in AML. It has been demonstrated that the hypoxic BMM creates a protective niche that mediates resistance to therapy. Therefore, we also highlight how hypoxia hallmarks might be targeted in the future to hit the leukemic population to improve AML patient outcomes.

Context: Histone H3 lysine 36 trimethylation (H3K36me3) by SETD2 is implicated in transcriptional regulation, splicing fidelity, and DNA damage response signaling. SETD2 non-genomic loss of function, due to proteasome-mediated protein degradation, is a common event in advanced systemic mastocytosis (advSM). Objective: To investigate the mechanisms underlying SETD2 loss of function in advSM to highlight possible druggable targets. Patients or other participants: DNA and protein samples from a retrospective cohort of 88 patients with systemic mastocytosis, classified according to WHO criteria were used for sequencing, western blotting (WB), and immunoprecipitation studies. Neoplastic mast cells (MCs) from 10 pts with advSM were used for ex-vivo drug testing. Results: Aurora kinase A (AKA) was overexpressed and hyper-activated in advSM and this inversely correlated with SETD2 protein expression. siRNA-mediated silencing of AKA and pharmacological inhibition by danusertib rescued SETD2 expression and activity, suggesting that AKA is implicated in SETD2 degradation. The new gold standard of therapy in advSM is midostaurin that targets not only mutant KIT but also other kinases, including AKA. To investigate whether midostaurin treatment may result in efficient AKA inhibition and consequent SETD2/H3K36me3 rescue, the HMC-1 cell line was treated with 5 μM midostaurin for 24 h and phospho-AKA (T288), SETD2, and H3K36me3 expression were evaluated by WB. Midostaurin reduced AKA phosphorylation by 60%, partially restoring SETD2 expression and activity, but did not induce cytotoxic effects. We next tested the efficacy of combined treatment with midostaurin and nilotinib or dasatinib in inducing cytotoxic rather than cytostatic effects. Cytofluorimetric analysis of apoptosis and clonogenic assays in HMC-1 cells and in neoplastic MCs showed an important advantage in using the midostaurin + dasatinib/nilotinib combination compared to each single agent alone, as underlined by the significant reduction of drug doses necessary to obtain cytotoxic effects and cell growth arrest. Of note, we observed that midostaurin + nilotinib completely de-phosphorylated AKA and restored SETD2 expression and activity. Conclusions: AKA overexpression contributes to SETD2 non-genomic loss of function in advSM. KIT and AKA targeting by midostaurin in combination with second-generation tyrosine kinase inhibitors is a promising therapeutic alternative in patients with SETD2/H3K36me3 deficiency. Supported by AIRC project 23001.