Phosphoproteome and drug-response effects mediated by the three protein phosphatase 2A inhibitor proteins CIP2A, SET, and PME-1.

Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phosphotargets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A) (to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate nonredundancy among CIP2A, PME-1, and SET in phosphotarget regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of nonoverlapping phosphatase inhibition- and activation-responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies.

Breeze: an integrated quality control and data analysis application for high-throughput drug screening.

SUMMARY: High-throughput screening (HTS) enables systematic testing of thousands of chemical compounds for potential use as investigational and therapeutic agents. HTS experiments are often conducted in multi-well plates that inherently bear technical and experimental sources of error. Thus, HTS data processing requires the use of robust quality control procedures before analysis and interpretation. Here, we have implemented an open-source analysis application, Breeze, an integrated quality control and data analysis application for HTS data. Furthermore, Breeze enables a reliable way to identify individual drug sensitivity and resistance patterns in cell lines or patient-derived samples for functional precision medicine applications. The Breeze application provides a complete solution for data quality assessment, dose-response curve fitting and quantification of the drug responses along with interactive visualization of the results. AVAILABILITY AND IMPLEMENTATION: The Breeze application with video tutorial and technical documentation is accessible at https://breeze.fimm.fi; the R source code is publicly available at https://github.com/potdarswapnil/Breeze under GNU General Public License v3.0. CONTACT: swapnil.potdar@helsinki.fi. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

JAK1/2 and BCL2 inhibitors synergize to counteract bone marrow stromal cell-induced protection of AML.

The bone marrow (BM) provides a protective microenvironment to support the survival of leukemic cells and influence their response to therapeutic agents. In acute myeloid leukemia (AML), the high rate of relapse may in part be a result of the inability of current treatment to effectively overcome the protective influence of the BM niche. To better understand the effect of the BM microenvironment on drug responses in AML, we conducted a comprehensive evaluation of 304 inhibitors, including approved and investigational agents, comparing ex vivo responses of primary AML cells in BM stroma-derived and standard culture conditions. In the stroma-based conditions, the AML patient cells exhibited significantly reduced sensitivity to 12% of the tested compounds, including topoisomerase II, B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2), and many tyrosine kinase inhibitors (TKIs). The loss of TKI sensitivity was most pronounced in patient samples harboring FLT3 or PDGFRB alterations. In contrast, the stroma-derived conditions enhanced sensitivity to Janus kinase (JAK) inhibitors. Increased cell viability and resistance to specific drug classes in the BM stroma-derived conditions was a result of activation of alternative signaling pathways mediated by factors secreted by BM stromal cells and involved a switch from BCL2 to BCLXL-dependent cell survival. Moreover, the JAK1/2 inhibitor ruxolitinib restored sensitivity to the BCL2 inhibitor venetoclax in AML patient cells ex vivo in different model systems and in vivo in an AML xenograft mouse model. These findings highlight the potential of JAK inhibitors to counteract stroma-induced resistance to BCL2 inhibitors in AML.

Systematic drug sensitivity testing reveals synergistic growth inhibition by dasatinib or mTOR inhibitors with paclitaxel in ovarian granulosa cell tumor cells.

OBJECTIVE: Resistance to standard chemotherapy poses a major clinical problem in the treatment of ovarian cancer patients. Adult-type granulosa cell tumor (AGCT) is a unique ovarian cancer subtype for which efficient treatment options are lacking in advanced disease. To this end, systematic drug response and transcriptomics profiling were performed to uncover new therapy options for AGCTs. METHODS: The responses of three primary and four recurrent AGCTs to 230 anticancer compounds were screened in vitro using a systematic drug sensitivity and resistance testing (DSRT) platform, coupled with mRNA sequencing. The responses of the AGCTs were compared with those of human granulosa luteal cells and bone marrow mononuclear cells. RESULTS: Patient-derived AGCT cells showed selective sensitivity to the Src family tyrosine kinase inhibitor dasatinib. A combination of either dasatinib or an mTOR-inhibitor everolimus with paclitaxel resulted in synergistic inhibition of AGCT cell viability. The key kinase targets of dasatinib and members of the mTOR pathway were constantly expressed at mRNA and protein levels, indicating multikinase signal addictions in the AGCT cells. Transcriptomic characterization of the tumors revealed no known oncogenic mutations, suggesting that the drug sensitivity of AGCTs was rather conveyed by selective target expression. CONCLUSIONS: We used a systematic functional approach to reveal novel treatment options for a unique gynecological cancer. The selective synergy found between taxanes and dasatinib or mTOR inhibitors warrants further clinical investigations of these combinations in relapsed or aggressive AGCTs and demonstrate that high-throughput drug screening and molecular profiling can provide an effective approach to uncover new therapy options.

Identification of selective cytotoxic and synthetic lethal drug responses in triple negative breast cancer cells.

BACKGROUND: Triple negative breast cancer (TNBC) is a highly heterogeneous and aggressive type of cancer that lacks effective targeted therapy. Despite detailed molecular profiling, no targeted therapy has been established. Hence, with the aim of gaining deeper understanding of the functional differences of TNBC subtypes and how that may relate to potential novel therapeutic strategies, we studied comprehensive anticancer-agent responses among a panel of TNBC cell lines. METHOD: The responses of 301 approved and investigational oncology compounds were measured in 16 TNBC cell lines applying a functional profiling approach. To go beyond the standard drug viability effect profiling, which has been used in most chemosensitivity studies, we utilized a multiplexed readout for both cell viability and cytotoxicity, allowing us to differentiate between cytostatic and cytotoxic responses. RESULTS: Our approach revealed that most single-agent anti-cancer compounds that showed activity for the viability readout had no or little cytotoxic effects. Major compound classes that exhibited this type of response included anti-mitotics, mTOR, CDK, and metabolic inhibitors, as well as many agents selectively inhibiting oncogene-activated pathways. However, within the broad viability-acting classes of compounds, there were often subsets of cell lines that responded by cell death, suggesting that these cells are particularly vulnerable to the tested substance. In those cases we could identify differential levels of protein markers associated with cytotoxic responses. For example, PAI-1, MAPK phosphatase and Notch-3 levels associated with cytotoxic responses to mitotic and proteasome inhibitors, suggesting that these might serve as markers of response also in clinical settings. Furthermore, the cytotoxicity readout highlighted selective synergistic and synthetic lethal drug combinations that were missed by the cell viability readouts. For instance, the MEK inhibitor trametinib synergized with PARP inhibitors. Similarly, combination of two non-cytotoxic compounds, the rapamycin analog everolimus and an ATP-competitive mTOR inhibitor dactolisib, showed synthetic lethality in several mTOR-addicted cell lines. CONCLUSIONS: Taken together, by studying the combination of cytotoxic and cytostatic drug responses, we identified a deeper spectrum of cellular responses both to single agents and combinations that may be highly relevant for identifying precision medicine approaches in TNBC as well as in other types of cancers.

Target inhibition networks: predicting selective combinations of druggable targets to block cancer survival pathways.

A recent trend in drug development is to identify drug combinations or multi-target agents that effectively modify multiple nodes of disease-associated networks. Such polypharmacological effects may reduce the risk of emerging drug resistance by means of attacking the disease networks through synergistic and synthetic lethal interactions. However, due to the exponentially increasing number of potential drug and target combinations, systematic approaches are needed for prioritizing the most potent multi-target alternatives on a global network level. We took a functional systems pharmacology approach toward the identification of selective target combinations for specific cancer cells by combining large-scale screening data on drug treatment efficacies and drug-target binding affinities. Our model-based prediction approach, named TIMMA, takes advantage of the polypharmacological effects of drugs and infers combinatorial drug efficacies through system-level target inhibition networks. Case studies in MCF-7 and MDA-MB-231 breast cancer and BxPC-3 pancreatic cancer cells demonstrated how the target inhibition modeling allows systematic exploration of functional interactions between drugs and their targets to maximally inhibit multiple survival pathways in a given cancer type. The TIMMA prediction results were experimentally validated by means of systematic siRNA-mediated silencing of the selected targets and their pairwise combinations, showing increased ability to identify not only such druggable kinase targets that are essential for cancer survival either individually or in combination, but also synergistic interactions indicative of non-additive drug efficacies. These system-level analyses were enabled by a novel model construction method utilizing maximization and minimization rules, as well as a model selection algorithm based on sequential forward floating search. Compared with an existing computational solution, TIMMA showed both enhanced prediction accuracies in cross validation as well as significant reduction in computation times. Such cost-effective computational-experimental design strategies have the potential to greatly speed-up the drug testing efforts by prioritizing those interventions and interactions warranting further study in individual cancer cases.

Mechanical forces across compartments coordinate cell shape and fate transitions to generate tissue architecture.

Morphogenesis and cell state transitions must be coordinated in time and space to produce a functional tissue. An excellent paradigm to understand the coupling of these processes is mammalian hair follicle development, which is initiated by the formation of an epithelial invagination-termed placode-that coincides with the emergence of a designated hair follicle stem cell population. The mechanisms directing the deformation of the epithelium, cell state transitions and physical compartmentalization of the placode are unknown. Here we identify a key role for coordinated mechanical forces stemming from contractile, proliferative and proteolytic activities across the epithelial and mesenchymal compartments in generating the placode structure. A ring of fibroblast cells gradually wraps around the placode cells to generate centripetal contractile forces, which, in collaboration with polarized epithelial myosin activity, promote elongation and local tissue thickening. These mechanical stresses further enhance compartmentalization of Sox9 expression to promote stem cell positioning. Subsequently, proteolytic remodelling locally softens the basement membrane to facilitate a release of pressure on the placode, enabling localized cell divisions, tissue fluidification and epithelial invagination into the underlying mesenchyme. Together, our experiments and modelling identify dynamic cell shape transformations and tissue-scale mechanical cooperation as key factors for orchestrating organ formation.

Obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection.

Influenza A viruses (IAVs) infect humans and cause significant morbidity and mortality. Different treatment options have been developed; however, these were insufficient during recent IAV outbreaks. Here, we conducted a targeted chemical screen in human nonmalignant cells to validate known and search for novel host-directed antivirals. The screen validated saliphenylhalamide (SaliPhe) and identified two novel anti-IAV agents, obatoclax and gemcitabine. Further experiments demonstrated that Mcl-1 (target of obatoclax) provides a novel host target for IAV treatment. Moreover, we showed that obatoclax and SaliPhe inhibited IAV uptake and gemcitabine suppressed viral RNA transcription and replication. These compounds possess broad spectrum antiviral activity, although their antiviral efficacies were virus-, cell type-, and species-specific. Altogether, our results suggest that phase II obatoclax, investigational SaliPhe, and FDA/EMEA-approved gemcitabine represent potent antiviral agents.

Targeting Apoptosis Pathways With BCL2 and MDM2 Inhibitors in Adult B-cell Acute Lymphoblastic Leukemia.

In adult patients, the treatment outcome of acute lymphoblastic leukemia (ALL) remains suboptimal. Here, we used an ex vivo drug testing platform and comprehensive molecular profiling to discover new drug candidates for B-ALL. We analyzed sensitivity of 18 primary B-ALL adult patient samples to 64 drugs in a physiological concentration range. Whole-transcriptome sequencing and publicly available expression data were used to examine gene expression biomarkers for observed drug responses. Apoptotic modulators targeting BCL2 and MDM2 were highly effective. Philadelphia chromosome-negative (Ph-) samples were sensitive to both BCL2/BCL-W/BCL-XL-targeting agent navitoclax and BCL2-selective venetoclax, whereas Ph-positive (Ph+) samples were more sensitive to navitoclax. Expression of BCL2 was downregulated and BCL-W and BCL-XL upregulated in Ph+ ALL compared with Ph- samples, providing elucidation for the observed difference in drug responses. A majority of the samples were sensitive to MDM2 inhibitor idasanutlin. The regulatory protein MDM2 suppresses the function of tumor suppressor p53, leading to impaired apoptosis. In B-ALL, the expression of MDM2 was increased compared with other hematological malignancies. In B-ALL cell lines, a combination of BCL2 and MDM2 inhibitor was synergistic. In summary, antiapoptotic proteins including BCL2 and MDM2 comprise promising targets for future drug studies in B-ALL.

IFN-α with dasatinib broadens the immune repertoire in patients with chronic-phase chronic myeloid leukemia.

In chronic myeloid leukemia (CML), combination therapies with tyrosine kinase inhibitors (TKIs) aim to improve the achievement of deep molecular remission that would allow therapy discontinuation. IFN-α is one promising candidate, as it has long-lasting effects on both malignant and immune cells. In connection with a multicenter clinical trial combining dasatinib with IFN-α in 40 patients with chronic-phase CML (NordCML007, NCT01725204), we performed immune monitoring with single-cell RNA and T cell receptor (TCR) sequencing (n = 4, 12 samples), bulk TCRß sequencing (n = 13, 26 samples), flow cytometry (n = 40, 106 samples), cytokine analyses (n = 17, 80 samples), and ex vivo functional studies (n = 39, 80 samples). Dasatinib drove the immune repertoire toward terminally differentiated NK and CD8+ T cells with dampened functional capabilities. Patients with dasatinib-associated pleural effusions had increased numbers of CD8+ recently activated effector memory T (Temra) cells. In vitro, dasatinib prevented CD3-induced cell death by blocking TCR signaling. The addition of IFN-α reversed the terminally differentiated phenotypes and increased the number of costimulatory intercellular interactions and the number of unique putative epitope-specific TCR clusters. In vitro IFN-α had costimulatory effects on TCR signaling. Our work supports the combination of IFN-α with TKI therapy, as IFN-α broadens the immune repertoire and restores immunological function.

Anti-COX-2 autoantibody is a novel biomarker of immune aplastic anemia.

In immune aplastic anemia (IAA), severe pancytopenia results from the immune-mediated destruction of hematopoietic stem cells. Several autoantibodies have been reported, but no clinically applicable autoantibody tests are available for IAA. We screened autoantibodies using a microarray containing >9000 proteins and validated the findings in a large international cohort of IAA patients (n = 405) and controls (n = 815). We identified a novel autoantibody that binds to the C-terminal end of cyclooxygenase 2 (COX-2, aCOX-2 Ab). In total, 37% of all adult IAA patients tested positive for aCOX-2 Ab, while only 1.7% of the controls were aCOX-2 Ab positive. Sporadic non-IAA aCOX-2 Ab positive cases were observed among patients with related bone marrow failure diseases, multiple sclerosis, and type I diabetes, whereas no aCOX-2 Ab seropositivity was detected in the healthy controls, in patients with non-autoinflammatory diseases or rheumatoid arthritis. In IAA, anti-COX-2 Ab positivity correlated with age and the HLA-DRB1*15:01 genotype. 83% of the >40 years old IAA patients with HLA-DRB1*15:01 were anti-COX-2 Ab positive, indicating an excellent sensitivity in this group. aCOX-2 Ab positive IAA patients also presented lower platelet counts. Our results suggest that aCOX-2 Ab defines a distinct subgroup of IAA and may serve as a valuable disease biomarker.

Implementing a Functional Precision Medicine Tumor Board for Acute Myeloid Leukemia.

We generated ex vivo drug-response and multiomics profiling data for a prospective series of 252 samples from 186 patients with acute myeloid leukemia (AML). A functional precision medicine tumor board (FPMTB) integrated clinical, molecular, and functional data for application in clinical treatment decisions. Actionable drugs were found for 97% of patients with AML, and the recommendations were clinically implemented in 37 relapsed or refractory patients. We report a 59% objective response rate for the individually tailored therapies, including 13 complete responses, as well as bridging five patients with AML to allogeneic hematopoietic stem cell transplantation. Data integration across all cases enabled the identification of drug response biomarkers, such as the association of IL15 overexpression with resistance to FLT3 inhibitors. Integration of molecular profiling and large-scale drug response data across many patients will enable continuous improvement of the FPMTB recommendations, providing a paradigm for individualized implementation of functional precision cancer medicine. SIGNIFICANCE: Oncogenomics data can guide clinical treatment decisions, but often such data are neither actionable nor predictive. Functional ex vivo drug testing contributes significant additional, clinically actionable therapeutic insights for individual patients with AML. Such data can be generated in four days, enabling rapid translation through FPMTB.See related commentary by Letai, p. 290.This article is highlighted in the In This Issue feature, p. 275.

Evaluation of hematological, coagulation and inflammatory biomarker's role in predicting the severity of disease in patients with COVID-19, admitted in designated COVID-19 hospital of central India.

BACKGROUND: COVID-19 is a pandemic viral disease that has affected the Indian population very badly with more than 8.46 million cases and > 0.125 million deaths. AIM: Primary objective of the study is to establish the role of hematological, coagulation and inflammatory biomarkers in early identification of clinically severe covid-19 cases. MATERIALS AND METHODS: This study was conducted from July 2020 to August 2020 at a dedicated COVID-19 referral hospital in central India. Only RT-PCR confirmed COVID-19 positive 300 cases admitted in the hospital were included in this study. Based on the clinical assessment, patients were categorised as mild, moderate, and severe groups as per ICMR guidelines. Blood samples of all cases were tested for haematological, coagulation and inflammatory biomarkers and mean values were compared among the three groups of patients. RESULTS: 46% patients belonged to >60 years of age group. Hematological parameters like total leukocyte count, absolute neutrophil count, Neutrophil: Lymphocyte ratio, Platelet: Lymphocyte ratio significantly increased with lymphocytopenia (P=0.001). Coagulation profile(D-dimer and PT) and inflammatory biomarkers like CRP, LDH, ferritin, procalcitonin and NT- Pro BNP, all were significantly increased with severity of patients(p=0.001). ROC plotted for all the parameters between severe v/s non-severe cases showed that CRP, LDH and D-dimer had a good discriminative precision with AUC >0.8. CONCLUSION: We suggest that biochemical markers like CRP, LDH and D-dimer can be used as a screening tool to differentiate severe patients from non-severe patients of Covid-19 disease in order to identify severe disease at early stage for optimal utilization of resources & reducing further morbidity & mortality.

High miR-30 Expression Associates with Improved Breast Cancer Patient Survival and Treatment Outcome.

Deregulated miRNA expression has been suggested in several stages of breast cancer pathogenesis. We have studied the miR-30 family, in particular miR-30d, in relation to breast cancer patient survival and treatment outcomes. With tumor specimens from 1238 breast cancer patients, we analyzed the association of miR-30d expression with tumor characteristics with the 5-year occurrence of breast cancer-specific death or distant metastasis (BDDM), and with 10-year breast cancer survival (BCS). We conducted a two-stage drug-screen to investigate the impact of miR-30 family members (miR-30a-30e) on sensitivity to doxorubicin and lapatinib in six breast cancer cell lines HCC1937, HCC1954, MDA-MB-361, MCF7, MDA-MB-436 and CAL-120, using drug sensitivity scores (DSS) to compare the miR-30 family mimics to their specific inhibitors. The study was complemented with Ingenuity Pathway Analysis (IPA) with the METABRIC data. We found that while high miR-30d expression is typical for aggressive tumors, it predicts better metastasis-free (pBDDM = 0.035, HR = 0.63, 95% CI = 0.4-0.9) and breast cancer-specific survival (pBCS = 0.018, HR = 0.61, 95% CI = 0.4-0.9), especially in HER2-positive (pBDDM = 0.0009), ER-negative (pBDDM = 0.003), p53-positive (pBDDM = 0.011), and highly proliferating (pBDDM = 0.0004) subgroups, and after adjuvant chemotherapy (pBDDM = 0.035). MiR-30d predicted survival independently of standard prognostic markers (pBDDM = 0.0004). In the drug-screening test, the miR-30 family sensitized the HER2-positive HCC1954 cell line to lapatinib (p < 10-2) and HCC1937, MDA-MB-361, MDA-MB-436 and CAL120 to doxorubicin (p < 10-4) with an opposite impact on MCF7. According to the pathway analysis, the miR-30 family has a suppressive effect on cell motility and metastasis in breast cancer. Our results suggest prognostic and predictive potential for the miR-30 family, which warrants further investigation.

Corrigendum: Adult-Onset Anti-Citrullinated Peptide Antibody-Negative Destructive Rheumatoid Arthritis Is Characterized by a Disease-Specific CD8+ T Lymphocyte Signature.

[This corrects the article DOI: 10.3389/fimmu.2020.578848.].

Adult-Onset Anti-Citrullinated Peptide Antibody-Negative Destructive Rheumatoid Arthritis Is Characterized by a Disease-Specific CD8+ T Lymphocyte Signature.

Rheumatoid arthritis (RA) is a complex autoimmune disease targeting synovial joints. Traditionally, RA is divided into seropositive (SP) and seronegative (SN) disease forms, the latter consisting of an array of unrelated diseases with joint involvement. Recently, we described a severe form of SN-RA that associates with characteristic joint destruction. Here, we sought biological characteristics to differentiate this rare but aggressive anti-citrullinated peptide antibody-negative destructive RA (CND-RA) from early seropositive (SP-RA) and seronegative rheumatoid arthritis (SN-RA). We also aimed to study cytotoxic CD8+ lymphocytes in autoimmune arthritis. CND-RA, SP-RA and SN-RA were compared to healthy controls to reveal differences in T-cell receptor beta (TCRß) repertoire, cytokine levels and autoantibody repertoires. Whole-exome sequencing (WES) followed by single-cell RNA-sequencing (sc-RNA-seq) was performed to study somatic mutations in a clonally expanded CD8+ lymphocyte population in an index patient. A unique TCRß signature was detected in CND-RA patients. In addition, CND-RA patients expressed higher levels of the bone destruction-associated TNFSF14 cytokine. Blood IgG repertoire from CND-RA patients recognized fewer endogenous proteins than SP-RA patients' repertoires. Using WES, we detected a stable mutation profile in the clonally expanded CD8+ T-cell population characterized by cytotoxic gene expression signature discovered by sc-RNA-sequencing. Our results identify CND-RA as an independent RA subset and reveal a CND-RA specific TCR signature in the CD8+ lymphocytes. Improved classification of seronegative RA patients underlines the heterogeneity of RA and also, facilitates development of improved therapeutic options for the treatment resistant patients.

Integrated Analysis of Drug Sensitivity and Selectivity to Predict Synergistic Drug Combinations and Target Coaddictions in Cancer.

High-throughput drug sensitivity testing provides a powerful phenotypic profiling approach to identify effective drug candidates for individual cell lines or patient-derived samples. Here, we describe an experimental-computational pipeline, named target addiction scoring (TAS), which mathematically transforms the drug response profiles into target addiction signatures, and thereby provides a ranking of potential therapeutic targets according to their functional importance in a particular cancer sample. The TAS pipeline makes use of drug polypharmacology to integrate the drug sensitivity and selectivity profiles through systems-wide interconnection networks between drugs and their targets, including both primary protein targets as well as secondary off-targets. We show how the TAS pipeline enables one to identify not only single-target addictions but also combinatorial coaddictions among targets that often underlie synergistic drug combinations.

Dasatinib and navitoclax act synergistically to target NUP98-NSD1+/FLT3-ITD+ acute myeloid leukemia.

Acute myeloid leukemia (AML) with co-occurring NUP98-NSD1 and FLT3-ITD is associated with unfavorable prognosis and represents a particularly challenging treatment group. To identify novel effective therapies for this AML subtype, we screened patient cells and engineered cell models with over 300 compounds. We found that mouse hematopoietic progenitors co-expressing NUP98-NSD1 and FLT3-ITD had significantly increased sensitivity to FLT3 and MEK-inhibitors compared to cells expressing either aberration alone (P < 0.001). The cells expressing NUP98-NSD1 alone had significantly increased sensitivity to BCL2-inhibitors (P = 0.029). Furthermore, NUP98-NSD1+/FLT3-ITD+ patient cells were also very sensitive to BCL2-inhibitor navitoclax, although the highest select sensitivity was found to SRC/ABL-inhibitor dasatinib (mean IC50 = 2.2 nM). Topoisomerase inhibitor mitoxantrone was the least effective drug against NUP98-NSD1+/FLT3-ITD+ AML cells. Of the 25 significant hits, four remained significant also compared to NUP98-NSD1-/FLT3-ITD+ AML patients. We found that SRC/ABL-inhibitor dasatinib is highly synergistic with BCL2-inhibitor navitoclax in NUP98-NSD1+/FLT3-ITD+ cells. Gene expression analysis supported the potential relevance of dasatinib and navitoclax by revealing significantly higher expression of BCL2A1, FGR, and LCK in NUP98-NSD1+/FLT3-ITD+ patients compared to healthy CD34+ cells. Our data suggest that dasatinib-navitoclax combination may offer a clinically relevant treatment strategy for AML with NUP98-NSD1 and concomitant FLT3-ITD.