Tumor-associated neutrophils (TANs) are abundant across cancers, yet their phenotypic diversity and functional states remain poorly defined. Here, we introduce a cell-type probability classifier that recovers low-transcript neutrophils from scRNAseq datasets, enabling pan-cancer analyses of TAN heterogeneity. Across >190 human and murine tumors, we identify a conserved differentiation trajectory that culminates in a terminal CCL3hi state. This state exhibits pro-tumor transcriptional programs, including those involved in hypoxic adaptation and senescence. Consistently, CCL3hi TANs are enriched in hypoxic tumor niches in both humans and mice. Through mechanistic perturbations of neutrophil-derived CCL3 in mice, we show that it sustains TAN survival in hypoxic tumor regions via CCR1-dependent signaling. These findings establish CCL3 as a conserved marker and functional driver of pro-tumor neutrophils in growing tumors, and provide a scalable framework for dissecting neutrophil biology across cancer types.

Enzyme replacement therapy (ERT) for Gaucher disease (GD) effectively prevents skeletal, visceral, and hematologic complications of this inherited, lysosomal storage disorder. Taliglucerase is one of the three commercially available ERT products and became the recommended first-line therapy in Québec, Canada in 2016. Thus, 19 patients were switched from imiglucerase to taliglucerase, but more than a quarter experienced significant side effects. Here, we summarize these patients' clinical course and describe 6 suspected product-related adverse-effects.

INTRODUCTION Co-mutations of BCR::ABL1 and clonal hematopoiesis genes are known to occur in chronic myeloid leukemia (CM)L, but the presence of mutant SF3B1 and BCR::ABL1 is rarely described. A 74-year-old man was referred for a hemoglobin of 91g/L with high MCV (122 fL); platelet and leucocyte counts were normal, as was white count differential. The patient had treated metastatic prostate cancer with good disease control. Bone marrow examination revealed hypercellularity (80%), erythroid predominance, ringed sideroblasts, <1% myeloblasts, and absence of basophilia. A t(9;22) was identified in 18/25 metaphases; molecular testing confirmed the presence of the p210 BCR::ABL1, IS 17.6%. A 36-gene myeloid next-generation sequencing panel revealed a missense mutation p.K666R in SF3B1 (VAF 46%). A diagnosis of concurrent CML and myelodysplastic syndrome (MDS) with mutated SF3B1 was made. Bone marrow sampling repeated 3 months after starting asciminib showed minimal change in cellularity and erythroid predominance. At this time, BCR::ABL1 dropped to 0.0063% IS and SF3B1 VAF was 32%. After a transient improvement in anemia and macrocytosis at 6 months, the hemoglobin dropped to 81, and MCV rose to 116 after 1 year, at which time BCR::ABL1 was undetectable. We performed single cell DNA sequencing to elucidate the relationship between the somatic SF3B1mutation and BCR::ABL1. METHODS Whole genome sequencing using Nanopore was used to identify the t(9;22) breakpoint. Probes were subsequently designed by Mission Bio to include the BCR::ABL1 translocation and the SF3B1 mutation. Using the Tapestri single-cell DNA sequencing platform, cells were isolated into individual droplets where they were lysed, amplified, tagged and sequenced. To obtain the lineage-specific genetic profile, cells were visualized using Uniform Manifold Approximation and Projection and clustered using Hierarchical Density-Based Spatial Clustering of Applications with Noise and manually gated. RESULTS A total of 4065 cells were sequenced. Most cells were erythroid precursors (88.8%, n=3609), followed by lymphocytes (8.45%, n=276), hematopoietic stem and progenitor cells (HSPC) (1.6%, n=65) and monocytes (0.86%, n=35). Among all sequenced cells, 45% (n=1779) harboured both mutant SF3B1 and BCR::ABL1; 25% (n=969) had mutant SF3B1 only. The remaining cells had neither abnormality. Within HSPC, 20% (n=13) were wild type, 24.6% (n=16) only had the SF3B1 mutation and 55.4% (n=36) had the SF3B1 mutation with BCR::ABL1. Erythroid precursors were 26.3% (n=949) wild type, 25.7% (n=926) only had the SF3B1 mutation and 48% (n=1734) had the SF3B1 mutation with BCR::ABL1. Among monocytes, 42.9% (n=15) were wild-type, 42.9% (n=15) had the SF3B1 mutation and 14.3% (n=5) had the SF3B1 mutation with BCR::ABL1. Finally, 97% of lymphocytes were wild type (n=269), 1.5% of lymphocytes only had the SF3B1 mutation (n=4) and 1.5% (n=3) had the SF3B1 mutation and BCR::ABL1.Interestingly, most of the erythroid precursors and HSPC contained the SF3B1 mutation, with or without BCR::ABL1. A smaller subset of monocytes contained both mutations and lymphocytes were mainly wild type. CONCLUSION In this unique case, the presence of mutated SF3B1 likely preceded the emergence BCR::ABL1 and exerted clinical and morphological dominance, yielding an MDS phenotype. At the single-cell level, mutant SF3B1 and BCR::ABL1 were notably absent from lymphoid cells. This contrasts with findings in CML, where BCR::ABL1 is usually present in all cell lineages at diagnosis (Haferlach, BJH, 1996). SF3B1 has been reported to impede lymphocyte differentiation (Mortera-Blanco, Blood, 2018) and seemingly also hampers myeloid proliferation typical of CML, as this case demonstrates. Granulocyte precursors likely experienced compromised viability during sample storage, leading to underrepresentation in the single-cell analysis. Nonetheless, a correlation persists between the reduced granulopoiesis observed in the bone marrow and the findings from single-cell analysis. Single-cell RNASeq is planned to delineate signaling pathways active in SF3B1 and BCR::ABL1 co-mutated cells. To our knowledge, this is the first report mapping the clonal architecture of a case of concurrent CML and SF3B1-mutated MDS using single-cell DNA sequencing. These findings not only provide novel insight into lineage restriction and clonal dominance but also reveal how a concurrent mutation can mask the classic disease phenotype of CML.

Mechanisms driving cell growth during clonal evolution in leukemia are not fully understood. We focused on epigenomic regulation of this process by analyzing the changes of chromatin marks and gene expression in independent leukemic clones evolving towards increased growth. The evolved subclones lost their growth differential ex vivo but restored it upon secondary transplantation, suggesting molecular memory of their evolutionary stage. Genome-wide, clonal evolution was associated with clone-specific gradual modulation of chromatin states and expression levels, with a surprising preferential trend of reversing the prior changes observed at the early leukemic stage. We leveraged clonal specificity of these modulation patterns to focus on the core gene set of potential growth regulators with consistent changes of expression and chromatin marks that were maintained in vivo and ex vivo in both independent clones. We selected three of these genes as candidates (Irx5 and Plag1 as growth suppressors and Smad1 as a driver) and validated their predicted growth effects by overexpression in leukemic subclones. AML patient data confirmed IRX5 and SMAD1 as markers of AML status in patients, suggesting that multiomic analysis of clonal evolution in a mouse model is a valuable predictive approach relevant to human AML.

Acute myeloid leukemia (AML) relapses in more than half of patients treated with chemotherapy and almost invariably develops resistance to targeted therapies. AML is characterized by dysregulated mTOR complex 1 (mTORC1) activity, which promotes the expression, at the transcriptional and translational levels, of drivers of cell growth, survival, and metabolic alterations. Eukaryotic initiation factor 4F (eIF4F), a complex under the control of mTORC1, directly regulates translation initiation of oncogenic mRNAs with structured 5’ untranslated regions (UTRs). eIF4A, the helicase of this complex, is of interest as a therapeutic target due to the recent development of pharmaceutical inhibitors. We hypothesized that eIF4A inhibition could abrogate the translation of mRNAs associated with metabolic rewiring and survival to therapy in AML cells.To study the impact of eIF4A inhibition on AML growth and response to therapy, we used cell line-derived and patient-derived xenotransplantation models, treated with eIF4A inhibitors of the rocaglate family (CR-1-31-B, 0.2 mg/kg/day I.P. for 7 days), as single agent or in combination with cytarabine or the BCL2 inhibitor venetoclax. In vivo, eIF4A inhibition has anti-leukemic activity as single agent and potentiates the effect of chemotherapy or BCL2 inhibition. Mechanistically, we found that eIF4A inhibition impairs glycolysis and oxidative phosphorylation, increases levels of reactive oxygen species, and decreases BCL2-family proteins. Pharmacological eIF4A inhibition is well tolerated, with minimal effects on normal myelopoiesis. Translatomic and metabolomic characterizations of eIF4A inhibition in these models is ongoing.Overall, we show in pre-clinical models that eIF4A inhibition combined with venetoclax or cytarabine has the potential to prevent the translation of mRNAs associated with therapeutic resistance.

For several decades, induction therapy with nucleoside analogs, in particular cytarabine (Ara-C) and, to a lesser extent, fludarabine, has been the standard of care for patients diagnosed with acute myeloid leukemia (AML). Still, the anti-tumor efficacy of nucleoside analogs is often limited by intrinsic and acquired drug resistance, thereby leading to poor therapeutic response and suboptimal clinical outcomes. Here, we used genome-wide CRISPR-based pharmacogenomic screening to map the genetic factors that modulate the response to nucleoside analogs in AML and identified the E3 ubiquitin ligase Herc1 as a key modulator of Ara-C response in the MLL/AF9 (MA) and the HOXA9/MEIS1 (HM) murine AML models both in vitro and in vivo. Loss of HERC1 enhanced nucleoside analog-induced cell death in both murine and human AML cell lines by compromising cell cycle progression. In-depth proteomic analysis and subsequent validation identified deoxycytidine kinase Dck as a novel target of Herc1 in MA and HM murine cells. We observed that HERC1 is overexpressed in AML compared to other cancer types and higher HERC1 expression is associated with shorter overall survival of patients with AML in the TCGA and BEAT-AML cohorts. Collectively, this study highlights the importance of HERC1 in the response of AML cells to nucleoside analogs, thereby establishing this E3 ubiquitin ligase as a novel predictive biomarker and potential therapeutic target for the treatment of AML.

Differentiation therapy has proven to be a success story for patients with acute promyelocytic leukemia. However, the remaining subtypes of acute myeloid leukemia (AML) are treated with cytotoxic chemotherapies that have limited efficacy and a high likelihood of resistance. As differentiation arrest is a hallmark of AML, there is increased interest in developing differentiation-inducing agents to enhance disease-free survival. Here, we provide a comprehensive review of current reports and future avenues of nucleic acid therapeutics for AML, focusing on the use of targeted nucleic acid drugs to promote differentiation. Specifically, we compare and discuss the precision of small interfering RNA, small activating RNA, antisense oligonucleotides, and aptamers to modulate gene expression patterns that drive leukemic cell differentiation. We delve into preclinical and clinical studies that demonstrate the efficacy of nucleic acid-based differentiation therapies to induce leukemic cell maturation and reduce disease burden. By directly influencing the expression of key genes involved in myeloid maturation, nucleic acid therapeutics hold the potential to induce the differentiation of leukemic cells towards a more mature and less aggressive phenotype. Furthermore, we discuss the most critical challenges associated with developing nucleic acid therapeutics for myeloid malignancies. By introducing the progress in the field and identifying future opportunities, we aim to highlight the power of nucleic acid therapeutics in reshaping the landscape of myeloid leukemia treatment.

Acute myeloid leukemia (AML) is the leading cause of adult leukemia deaths. Yet, many AML patients have no curative options or die immediately after intensive chemotherapy. Therefore, new treatment strategies are desperately needed. AML is characterized by the arrested differentiation and continued division of immature myeloid cells in the bone marrow. In a healthy state, myeloid cell differentiation is mediated by the master transcription factor C/EBPα encoded by the CEBPA gene. However, CEBPA is transcriptionally silenced in ∼30% of AML patients, leading to blocked differentiation. In fact, AML models show that increasing C/EBPα expression removes this differentiation block and halts leukemic cell division. Therefore, therapies that could specifically increase the expression of C/EBPα would be a promising AML treatment strategy. We hypothesize that RNA activation (RNAa) of CEBPA would reduce leukemic blast proliferation by inducing terminal differentiation, thus providing a new, less-toxic treatment option for these high-risk patients. To induce RNAa, we are using a chemically modified, short duplex RNA encapsulated into a liposomal nanoparticle, termed “MTL-CEBPA”. Here we report that MTL-CEBPA is selectively taken up by myeloid cells in vitro and in vivo and is functionally active once delivered. Specifically, MTL-CEBPA induces a >2-fold upregulation in CEBPA expression in AML cells. This CEBPA upregulation sensitizes AML cells to a commonly prescribed small molecule tyrosine kinase inhibitor, Gilteritinib, both in vitro and in a xenograft mouse model of human AML. Furthermore, MTL-CEBPA demonstrates an acceptable safety profile as reported in a recent phase Ib clinical trial for solid tumours (NCT02716012). Taken together, these results provide a proof-of-concept for the treatment of AML using RNAa.

Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. Recently, inhibition of BCL-2 with venetoclax in combination with hypomethylating agents has emerged as an attractive strategy for high-risk AML cases. Another promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1) (Oki et al, Nature Comm, 2021). However, clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. To curtail these drawbacks, we adopted a strategy of inhibiting an important effector of the mTORC1 signaling pathway controlling mRNA translation - the eukaryotic initiation factor 4A (eIF4A), subunit of the translation initiation complex eIF4F. Recent evidence suggests that translational programs mediated by the mTORC1/4E-BP/eIF4F axis can support resistance to therapy in various cancer models driven by oncogenic kinases, in part by allowing cellular metabolic plasticity (Hulea L et al, Cell Metab. 2018). In fact, metabolism, and specifically mitochondrial oxidative phosphorylation, has emerged as a central dependency of AML cells, sustaining resistance to therapy and recurrence. Using the MOLM-14 human AML cell line to model therapy-resistant disease, we previously demonstrated the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, both in vitro and in vivo (Fooks et al, J Exp Clin Cancer Res 2022). eIF4Ai affected cellular metabolism, by reducing mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Concomitantly, eIF4Ai decreased intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mitochondrial ROS. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Importantly, CR-1-31-B acted synergistically in vitro in combination with cytarabine or venetoclax. Recently, we have expanded our characterization of the eIF4Ai/venetoclax combination in MOML-14 cells, and showed that venetoclax potentiates the CR-1-31-B-induced inhibition of cellular respiration, glycolysis and ATP production (Seahorse). The combination provokes a robust apoptotic response with different temporal dynamics to single treatment, as measured using the Incucyte platform. In vivo, we have validated the strong anti-leukemic response induced by CR-1-31-B in a MOLM-14 transplantation model, after only 7 days of treatment, and showed that venetoclax slightly potentiates this effect. In addition, eIF4A inhibition reduces the levels of several metabolic proteins (GLS1, IDH1), which supports our previous observations of decreased levels of TCA cycle metabolites. We have confirmed the metabolic and pro-apoptotic effect of eIF4Ai in a second cellular model of AML, U937. Consistent with previous results, in U937 cells CR-1-31-B induces apoptosis at low nM concentrations, reduces cellular bioenergetics, ATP production, as well as levels of pro-proliferative (cyclin D3, CDK4) and anti-apoptotic (BCL-2, MCL-1) proteins. Our collective studies highlight (i) the importance of the crosstalk between mRNA translation and metabolic regulation and (ii) that direct inhibition of translation represents an appealing therapeutic strategy for clinical cases of therapy resistance that are dependent on the mTORC1/eIF4F axis. This is of interest as several translation inhibitors are currently tested in phase 1/2 clinical trials in solid malignancies. # co-corresponding authors

Gene therapy represents a significant potential to revolutionize the field of hematology with applications in correcting genetic mutations, generating cell lines and animal models, and improving the feasibility and efficacy of cancer immunotherapy. Compared to different genetic engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) emerged as an effective and versatile genetic editor with the ability to precisely modify the genome. The applications of genetic engineering in various hematological disorders have shown encouraging results. Monogenic hematological disorders can conceivably be corrected with single gene modification. Through the use of CRISPR-CAS9, restoration of functional red blood cells and hemostasis factors were successfully attained in sickle cell anemia, beta-thalassemia, and hemophilia disorders. Our understanding of hemato-oncology has been advanced via CRIPSR-CAS9 technology. CRISPR-CAS9 aided to build a platform of mutated genes responsible for cell survival and proliferation in leukemia. Therapeutic application of CRISPR-CAS9 when combined with chimeric antigen receptor (CAR) T cell therapy in multiple myeloma and acute lymphoblastic leukemia was feasible with attenuation of CAR T cell therapy pitfalls. Our review outlines the latest literature on the utilization of CRISPR-Cas9 in the treatment of beta-hemoglobinopathies and hemophilia disorders. We present the strategies that were employed and the findings of preclinical and clinical trials. Also, the review will discuss gene engineering in the field of hemato-oncology as a proper tool to facilitate and overcome the drawbacks of chimeric antigen receptor T cell therapy (CAR-T).

Myelodysplastic syndromes (MDSs) are a heterogenous group of diseases affecting the hematopoietic stem cell that are curable only by stem cell transplantation. Both hematopoietic cell intrinsic changes and extrinsic signals from the bone marrow (BM) niche seem to ultimately lead to MDS. Animal models of MDS indicate that alterations in specific mesenchymal progenitor subsets in the BM microenvironment can induce or select for abnormal hematopoietic cells. Here, we identify a subset of human BM mesenchymal cells marked by the expression of CD271, CD146, and CD106. This subset of human mesenchymal cells is comparable with mouse mesenchymal cells that, when perturbed, result in an MDS-like syndrome. Its transcriptional analysis identified Osteopontin (SPP1) as the most overexpressed gene. Selective depletion of Spp1 in the microenvironment of the mouse MDS model, Vav-driven Nup98-HoxD13, resulted in an accelerated progression as demonstrated by increased chimerism, higher mutant myeloid cell burden, and a more pronounced anemia when compared with that in wild-type microenvironment controls. These data indicate that molecular perturbations can occur in specific BM mesenchymal subsets of patients with MDS. However, the niche adaptations to dysplastic clones include Spp1 overexpression that can constrain disease fitness and potentially progression. Therefore, niche changes with malignant disease can also serve to protect the host.

Acute myeloid leukemia (AML) is a cancer that originates from the bone marrow (BM). Under physiological conditions, the bone marrow supports the homeostasis of immune cells and hosts memory lymphoid cells. In this review, we summarize our present understanding of the role of the immune microenvironment on healthy bone marrow and on the development of AML, with a focus on T cells and other lymphoid cells. The types and function of different immune cells involved in the AML microenvironment as well as their putative role in the onset of disease and response to treatment are presented. We also describe how the immune context predicts the response to immunotherapy in AML and how these therapies modulate the immune status of the bone marrow. Finally, we focus on allogeneic stem cell transplantation and summarize the current understanding of the immune environment in the post-transplant bone marrow, the factors associated with immune escape and relevant strategies to prevent and treat relapse.

Rationale: IL-17 is a family of six pro-inflammatory cytokines (named from A to F) with distinct patterns of cellular expression. Among these, IL-17A is secreted by lymphoid subsets such as T-helper 17 cells and innate lymphoid cells type 3. Through its action on many tissues, IL-17A is recognized as a key mediator of response to infection and pathogenic states such as cancer and auto-immunity. Until now, the main effect of IL-17A on hematopoiesis has been attributed to an indirect loop through IL-17RA signaling on bone marrow stromal cells, which stimulates secretion of hematopoietic factors such as G-CSF. However, we hypothesized that IL-17A can directly signal to hematopoietic stem and progenitor cells (HSPCs) to determine their self-renewal and differentiation. Methodology: We analyzed the expression of the IL-17 signaling pathway in publicly available human and mouse hematopoietic single-cell RNAseq datasets. Mouse HSPCs were isolated using FACS to perform clonogenic and differentiation assays in Methocult (StemCell Technologies) and StemPro-34 (Gibco). Spectral flow cytometry (SONY ID7000) was used for analysing IL-17RA expression and differentiation of mouse HSPCs. IL-17A levels were measured in blood and bone marrow serum using ELISA (Invitrogen). Results: IL-17RA is expressed at the surface of mouse HSPCs, with greatest levels in common lymphoid progenitors and granulocyte-monocyte progenitors. mRNA expression of IL-17RA signaling pathway is also detectable in human HSPCs, with greatest levels in young individuals. IL-17A supplementation leads to an increase in clonogenic capacity of mouse HSPCs in methylcellulose colony-formation assays. In liquid culture, supplementation with IL-17A leads to a greater expansion of sorted Lin -/s-kit +/Sca-1 + progenitors, accompanied by an increased proportion of myeloid-committed cells.IL-17A is also detected in the bone marrow serum, with possible production from subsets of cells expressing RORγt, a master regulator in development of T helper 17 (Th17) cells. Conclusions: Our work suggests the ability of IL-17A to directly promote clonogenic potential of HSPCs and drive a myeloid-biased lineage commitment in progenitor subsets. Additional studies are required to characterize the response of HSPCs to direct IL-17A signaling in vivo. Considering the important role of IL-17A as a mediator of immune response, and tissue repair, further studies are warranted to elucidate whether modulation of this pathway may be therapeutic in the context of hematopoietic failure or malignancy.

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive, uncurable blood cancer associated with poor therapeutic response and high mortality. We developed CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation, which occurs in human AMKL, increased the penetrance and decreased the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We uncover the dominant oncogenic properties of GLIS2, which trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of numerous BH3-only proteins, causing AMKL cell sensitivity to the BCL-2 inhibitor navitoclax both in vitro and in vivo , suggesting a novel therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL. Key points GLIS2 cooperates with activated Nras to promote the development of acute megakaryoblastic leukemia. CBFA2T3-GLIS2 and GLIS2 alter the expression of BCL2 family members rendering AMKL cells sensitive to navitoclax.

Immunologic memory is a feature typically ascribed to the adaptive arm of the immune system. However, recent studies have demonstrated that hematopoietic stem cells (HSCs) and innate immune cells such as monocytes and macrophages can gain epigenetic signatures to enhance their response in the context of reinfection. This suggests the presence of long-term memory, a phenomenon referred to as trained immunity. Trained immunity in HSCs can occur via changes in the epigenetic landscape and enhanced chromatin accessibility in lineagespecific genes, as well as through metabolic alterations. These changes can lead to a skewing in lineage bias, particularly enhanced myelopoiesis and the generation of epigenetically modified innate immune cells that provide better protection against pathogens on secondary infection. Here, we summarize recent advancements in trained immunity and epigenetic memory formation in HSCs and self-renewing alveolar macrophages, which was the focus of the Spring 2022 International Society for Experimental Hematology (ISEH) webinar. (c) 2023 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Previous chemotherapy research has focused almost exclusively on apoptosis. Here, a standard front-line drug combination of cytarabine and idarubicin induces distinct features of caspase-independent, poly (ADP-ribose) polymerase 1 (PARP-1)-mediated programmed cell death "parthanatos"in acute myeloid leukemia (AML) cell lines (n = 3/10 tested), peripheral blood mononuclear cells from healthy human donors (n = 10/10 tested), and primary cell samples from patients with AML (n = 18/39 tested, French-American-British subtypes M4 and M5). A 3-fold improvement in survival rates is observed in the parthanatos-positive versus-negative patient groups (hazard ratio [HR] = 0.28-0.37, p = 0.002- 0.046). Manipulation of PARP-1 activity in parthanatos-competent cells reveals higher drug sensitivity in cells that have basal PARP-1 levels as compared with those subjected to PARP-1 overexpression or suppression. The same trends are observed in RNA expression databases and support the conclusion that PARP-1 can have optimal levels for favorable chemotherapeutic responses.

Multi-omic approaches offer an unprecedented overview of the development, plasticity, and resistance of cancer. However, the translation from anti-cancer compounds identified in vitro to clinically active drugs have a notoriously low success rate. Here, we review how technical advances in cell culture, robotics, computational biology, and development of reporter systems have transformed drug discovery, enabling screening approaches tailored to clinically relevant functional readouts (e.g., bypassing drug resistance). Illustrating with selected examples of "success stories," we describe the process of phenotype-based high-throughput drug screening to target malignant cells or the immune system. Second, we describe computational approaches that link transcriptomic profiling of cancers with existing pharmaceutical compounds to accelerate drug repurposing. Finally, we review how CRISPR-based screening can be applied for the discovery of mechanisms of drug resistance and sensitization. Overall, we explore how the complementary strengths of each of these approaches allow them to transform the paradigm of pre-clinical drug development.

Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP(+) ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.