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Monosomy 7 and del(7q) (-7/-7q) are frequent chromosomal abnormalities detected in up to 10% of acute myeloid leukemia (AML) patients. Despite unfavorable treatment outcomes, no approved targeted therapies exist for patients with -7/-7q. Therefore, we aimed to identify novel therapeutic vulnerabilities in AML with -7/-7q. Through an analysis of data from ex vivo drug screens in 270 primary AML samples, we discovered that -7/-7q AML cells are highly sensitive to the inhibition of nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is a rate-limiting enzyme in the NAD+ salvage pathway. Mechanistically, theNAMPTgene is located at 7q22.1, and deletion of one copy due to -7/-7q results inNAMPThaploinsufficiency. This leads to reduced gene expression and a therapeutically targetable vulnerability to the inhibition of NAMPT. Our results show that in -7/-7q AML, differentiated CD34+CD38+ progenitor cells are the most sensitive to the inhibition of NAMPT. In addition, we found that the combination of BCL2 inhibitor venetoclax and a NAMPT inhibitor efficiently eradicated undifferentiated AML blasts with -7/-7q. In conclusion, our findings demonstrate that AML samples with -7/-7q are highly sensitive to NAMPT inhibition, suggesting that NAMPT inhibitors have the potential to be an effective targeted therapy for patients with monosomy 7 or del(7q).KEY POINTSMonosomy 7 and del(7q) result in a one-copy deletion of the NAMPT gene at 7q22.1NAMPT haploinsufficiency causes a vulnerability to the inhibition of NAMPT

Funding Information: This study was supported by the Cancer Foundation Finland, the Helsinki Institute of Life Science (HiLIFE) Fellow grants, the Academy of Finland (grant 1320185 ), the Sigrid Jusélius Foundation , the Finnish Cancer Institute , the Finnish Medical Foundation , University of Helsinki , and the Finnish special governmental subsidy for health sciences, research, and training . The FIMM High Throughput Biomedicine Unit is financially supported by the University of Helsinki (HiLIFE) and Biocenter Finland. O.D. was supported by the K. Albin Johansson Foundation , the Emil Aaltonen Foundation , and the Juhani Aho foundation . Publisher Copyright: © 2023 The American Society of Hematology Myeloid neoplasms with erythroid or megakaryocytic differentiation include pure erythroid leukemia, myelodysplastic syndrome with erythroid features, and acute megakaryoblastic leukemia (FAB M7) and are characterized by poor prognosis and limited treatment options. Here, we investigate the drug sensitivity landscape of these rare malignancies. We show that acute myeloid leukemia (AML) cells with erythroid or megakaryocytic differentiation depend on the antiapoptotic protein B-cell lymphoma (BCL)-XL, rather than BCL-2, using combined ex vivo drug sensitivity testing, genetic perturbation, and transcriptomic profiling. High-throughput screening of >500 compounds identified the BCL-XL–selective inhibitor A-1331852 and navitoclax as highly effective against erythroid/megakaryoblastic leukemia cell lines. In contrast, these AML subtypes were resistant to the BCL-2 inhibitor venetoclax, which is used clinically in the treatment of AML. Consistently, genome-scale CRISPR-Cas9 and RNAi screening data demonstrated the striking essentiality of BCL-XL-encoding BCL2L1 but not BCL2 or MCL1, for the survival of erythroid/megakaryoblastic leukemia cell lines. Single-cell and bulk transcriptomics of patient samples with erythroid and megakaryoblastic leukemias identified high BCL2L1 expression compared with other subtypes of AML and other hematological malignancies, where BCL2 and MCL1 were more prominent. BCL-XL inhibition effectively killed blasts in samples from patients with AML with erythroid or megakaryocytic differentiation ex vivo and reduced tumor burden in a mouse erythroleukemia xenograft model. Combining the BCL-XL inhibitor with the JAK inhibitor ruxolitinib showed synergistic and durable responses in cell lines. Our results suggest targeting BCL-XL as a potential therapy option in erythroid/megakaryoblastic leukemias and highlight an AML subgroup with potentially reduced sensitivity to venetoclax-based treatments.

Publisher Copyright: © 2022 Okoye-Okafor et al. Thrombocytopenia, prevalent in the majority of patients with myeloid malignancies, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), is an independent adverse prognostic factor. Azacitidine (AZA), a mainstay therapeutic agent for stem cell transplant–ineligible patients with MDS/AML, often transiently induces or further aggravates disease-associated thrombocytopenia by an unknown mechanism. Here, we uncover the critical role of an acute type-I interferon (IFN-I) signaling activation in suppressing megakaryopoiesis in AZA-mediated thrombocytopenia. We demonstrate that megakaryocytic lineage-primed progenitors present IFN-I receptors and, upon AZA exposure, engage STAT1/SOCS1-dependent downstream signaling prematurely attenuating thrombopoietin receptor (TPO-R) signaling and constraining megakaryocytic progenitor cell growth and differentiation following TPO-R stimulation. Our findings directly implicate RNA demethylation and IFN-I signal activation as a root cause for AZA-mediated thrombocytopenia and suggest mitigation of TPO-R inhibitory innate immune signaling as a suitable therapeutic strategy to support platelet production, particularly during the early phases of AZA therapy.

Thrombocytopenia defined as platelet counts of Several studies evaluating the use of thrombopoietin receptor agonists (TPO-RA) for the clinical management of thrombocytopenia have shown promising clinical results. TPO-RA eltrombopag (EP) which has been effective as a single agent to raise platelet counts in MDS, but failed to stimulate platelet production in a phase III placebo-controlled clinical study when used in combination with AZA (NCT02158936). Here, we assessed the molecular and cellular mechanisms of AZA contributing to thrombocytopenia and interfering with TPO-RA mediated rescue. Our results demonstrate that AZA mediates the rapid induction of dsRNAs and activation of interferon type I (IFN-I) signaling in various hematopoietic cells, including stem and progenitor cells of healthy donors and MDS/AML. This engagement of IFN-I/STAT1/SOCS1 signaling resulted in significant inhibition of megakaryocytic progenitor growth and differentiation, independently of DNA hypomethylation and endogenous retroviruses. We show that the inhibitory effects of AZA on megakaryopoeisis can be counteracted through inhibition of IFN-I signaling, SOCS1 activation or P38 MAPK activity. Our findings provide evidence intercepting inhibition of TPO-R signaling by AZA-induced innate immune IFN-I pathway and thrombocytopenia.

Drug combination therapy has the potential to enhance efficacy, reduce dose-dependent toxicity and prevent the emergence of drug resistance. However, discovery of synergistic and effective drug combinations has been a laborious and often serendipitous process. In recent years, identification of combination therapies has been accelerated due to the advances in high-throughput drug screening, but informatics approaches for systems-level data management and analysis are needed. To contribute toward this goal, we created an open-access data portal (https://drugcomb.fimm.fi) where the results of drug combination screening studies are accumulated, standardized and harmonized. Through the data portal, we provided the web server to analyze and visualize users’ own drug combination screening data. The users have an option to upload their data to DrugComb, as part of a crowdsourcing data curation effort. To initiate the data repository, we collected 437,932 drug combinations tested on a variety of cancer cell lines. We showed that linear regression approaches, when considering chemical fingerprints as predictors, have the potential to achieve high accuracy of predicting the sensitivity and synergy of drug combinations. All the data and informatics tools are freely available in DrugComb to enable a more efficient utilization of data resources for future drug combination discovery.

Drug combination therapy has the potential to enhance efficacy, reduce dose-dependent toxicity and prevent the emergence of drug resistance. However, discovery of synergistic and effective drug combinations has been a laborious and often serendipitous process. In recent years, identification of combination therapies has been accelerated due to the advances in high-throughput drug screening, but informatics approaches for systems-level data management and analysis are needed. To contribute toward this goal, we created an open-access data portal called DrugComb (https://drugcomb.fimm.fi) where the results of drug combination screening studies are accumulated, standardized and harmonized. Through the data portal, we provided a web server to analyze and visualize users’ own drug combination screening data. The users can also effectively participate a crowdsourcing data curation effect by depositing their data at DrugComb. To initiate the data repository, we collected 437 932 drug combinations tested on a variety of cancer cell lines. We showed that linear regression approaches, when considering chemical fingerprints as predictors, have the potential to achieve high accuracy of predicting the sensitivity of drug combinations. All the data and informatics tools are freely available in DrugComb to enable a more efficient utilization of data resources for future drug combination discovery.

The inverse dynamic simulation allows a designer to test the dynamic performance of a closed loop controlled drive without the need to parametrize a feedback controller. Equation based object oriented modeling languages such as Modelica are suitable to build models that can be simulated both, in a forward or inverse fashion. The same model can be used to simulate either in forward fashion or inversely, depending purely on which boundary conditions are specified. To evaluate the usefulness and limitations of the inverse simulation approach for the sizing of a hydraulic servo drive, an open source Modelica library OpenHydraulics is modified to enable inverse simulation. The library is then used for a case study about the sizing of a valve/cylinder hydraulic servo-drive. The inverse simulation supports an intuitive, iterative design approach towards the sizing problem of closed loop controlled drives, such as hydraulic or electric servo drives. The designer can change the size of system components and at any...

Management of hematologic disease- and therapy-related thrombocytopenia remains a serious clinical issue, especially in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The ribonucleoside and DNA-demethylating agent azacytidine (AZA), has proven useful for the treatment of patients with MDS and AML not eligible for stem cell transplantation. While low-dose AZA therapy induces clinical remissions in up to 50% of treated patients, it comes at the cost of aggravating pre-existing thrombocytopenia which is observed in a subset of patients; this can lead to increased bleeding and bleeding-associated mortality, and importantly, often requires dose modifications and delays of therapy. Thus, identification of strategies alleviating ineffective megakaryopoiesis will likely lead to increased therapeutic efficacy for patients with MDS/AML. Eltrombopag (EP), a second-generation small molecule thrombopoietin receptor (TPO-R) agonist was effective in raising platelet counts in patients with MDS as a single agent, as well as in combination with certain standard of care therapies. However, it failed to stimulate platelet production during the first four cycles of AZA treatment as uncovered by a recent phase III placebo-controlled clinical study (SUPPORT; NCT02158936). The goals of this study were to identify the cellular and molecular underpinnings of AZA-associated inhibition of megakaryopoiesis and to assess the ineffectiveness of EP in mitigating AZA treatment-associated thrombocytopenia. Our results demonstrate that at a clinically-equivalent and non-cytotoxic dose, AZA rapidly induces transient activation of interferon type I (IFN-I) signaling in various hematopoietic cell types, including stem and lineage-committed progenitor cells (HSPCs). We detected IFNα and IFNβ production and release using ELISA and intracellular flow cytometry on primary total mononuclear cell- and purified CD34-positive HSPC populations derived from cord blood, bone marrow from healthy volunteers or patients with MDS/AML. AZA-mediated activation of Type I IFNs in healthy control- and MDS/AML cells was preceded by an accumulation of double-stranded RNA (dsRNA) species and decreased total RNA cytosine methylation measured by immunocytochemistry and intracellular FACS analysis; this suggested that AZA triggered the accumulation of immunogenic RNA species which elicit an IFN-I response. In support, we found Toll like receptor 3 (TLR3) activation and phosphorylation of STAT1 in CD34+ HPSC, along with premature activation of Suppressor of Cytokine Signaling 1 (SOCS1), a well-known JAK/STAT-dependent signaling attenuator. This rapid AZA-induced viral mimicry response led to abrogation of thrombopoietin (TPO) or EP-stimulated TPO-R signaling and inhibition of ex vivo megakaryocyte progenitor proliferation quantified by colony formation in semi-solid medium. Importantly, inhibition of IFN-I signal activation using the JAK3 inhibitor decernotinib, the IFNα/β-blocking peptide, B18R, or RNA interference-mediated knock-down of SOCS1 counteracted the inhibitory effects of AZA on TPO-R stimulation and restored megakaryopoiesis. Given these observations, we pre-clinically tested a revised treatment protocol, in which primary cells were first exposed to AZA for four days followed by TPO-R stimulation using TPO or EP. This new treatment strategy alleviated AZA's inhibitory effects at the molecular and cellular levels, demonstrating that upon resolution of the AZA-mediated vial mimicry response, EP and TPO can effectively stimulate TPO-R signaling and megakaryopoiesis. Together, our data reveal a mechanistic basis of AZA-mediated inhibition of megakaryopoiesis in patients with MDS/AML. Additionally, we show that EP cannot overcome the megakaryopoiesis-inhibitory effects of acute IFN-I signaling activation upon AZA exposure. Findings of our study are consistent with and provide a molecular explanation for the observations made in the context of the SUPPORT study. In the future, it will be critical to better understand and potentially counteract the megakaryopoiesis-inhibitory effects by IFN-I pathway activation upon AZA therapy in patients with MDS/AML. Disclosures Okoye-Okafor: Novartis Pharmaceuticals: Research Funding. Pallaud:Novartis Pharmaceuticals: Employment. Marques Ramos:Novartis Pharmaceuticals: Employment. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria. Heckman:Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding; Orion Pharma: Research Funding. Will:Novartis Pharmaceuticals: Research Funding.

BACKGROUND: Despite mounting knowledge, the management of patients with acute myeloid leukemia (AML) can be challenging, particularly for relapsed/refractory patients who lack innovative, effective treatments. Recent preclinical studies of AML have demonstrated promissory therapeutic activity of the novel class of inhibitors of bromodomain and extra terminal (BET) family proteins. To identify AML patients that may benefit from BET inhibitor treatment, we sought to discover and evaluate predictive molecular biomarkers for sensitivity towards BET inhibition, focusing on the prototype BET inhibitor JQ1. METHODS: Following written informed consent and according to the Declaration of Helsinki, bone marrow, peripheral blood, and skin biopsy samples were obtained prospectively from AML patients (n=139) and analyzed for their whole exome profiles and ex vivo responses to JQ1 exposure, as part of an Individualized Systems Medicine (ISM) study of AML (Pemovska et al., 2013). Consequently, associations between molecular features and drug responses were investigated using logistic regression models for data integration. Novel signals emerging from our cohort of patients were carried forward for reproducibility assessment in publicly available cell line datasets (Cancer Cell Line Encyclopedia and Catalogue of Somatic Mutations in Cancer for genomic data, and Cancer Therapeutics Response Portal and Genomics of Drug Sensitivity in Cancer for drug response data). In addition, literature-reported indicators of sensitivity were examined in the AML cohort in order to evaluate their clinical translatability. RESULTS: Enabling a genome-wide search for predictive biomarkers for BET inhibitor response, our bioinformatics pipeline returned a set of signals significantly associated with JQ1 treatment. Frequently mutated in AML, isocitrate dehydrogenases IDH1 and IDH2, tet methylcytosine dioxygenase TET2, and Wilms' tumor WT1 each appeared to confer resistance upon mutation to the samples harboring them. Participating in a common biochemical pathway, the 4 genes have been shown to be mutated following a mutual exclusivity pattern in AML (Scourzic et al., 2015), which was successfully replicated in our cohort. Accordingly, grouping of samples with mutation in any of the genes yielded a significantly more resistant group to JQ1 treatment in comparison to wild-type samples. Incorporation of TET2 and WT1 deletions from the samples' copy number variation data supported this hypothesis. Importantly, this observation was also successfully validated in cell line drug sensitivity data (Figure 1A). Moreover, FLT3-mutated samples in our cohort returned significantly higher sensitivity scores to JQ1 compared to wild-type samples, irrespective of their NPM1 mutation status. The superior response trend observed within this group was maintained independently of whether the mutation was an internal tandem duplication (FLT3-ITD) or in the tyrosine kinase domain (FLT3-TKD) of the gene (Figure 1B). Finally, and in contrast to other studies, AML samples harboring mutation to NPM1 were not significantly more sensitive to BET inhibition with respect to NPM1 wild-type samples. CONCLUSION: Taken together, the results suggest novel insights for the stratification of AML patients for treatment using BET inhibition therapy, taking this malignancy's heterogeneity into account. Particularly, while FLT3 mutations are to be considered as sensitizing factors to BET inhibitors, samples with mutations in IDH1, IDH2, TET2, or WT1 were resistant, and thus, patients harboring these gene mutations may not benefit. These observations also propose drug partners that are worth investigating in combination with BET inhibitors in the defined patient subpopulations, namely FLT3 inhibitors and hypomethylating agents (or IDH1/2 inhibitors), respectively. Furthermore, the variability of responses observed among samples harboring NPM1 mutations highlights the molecular heterogeneity existing within this group of patients, stressing the need for identifying additional companion diagnostics for a more effective stratification of this population. Although our findings require additional validation in a larger cohort and through clinical investigations, this study provides evidence of molecular indicators that could help identify AML patients that would benefit from BET inhibitor therapy. Disclosures Tsallos: Novartis: Research Funding. Wennerberg:Novartis: Research Funding. Porkka:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Heckman:Novartis: Research Funding; Orion Pharma: Research Funding; Celgene: Research Funding.

Inverse dynamic simulation of hydraulic drives is helpful in early design stages of hydraulic machines to answer the question whether the drive can meet dynamic load requirements and at the same time to predict the energy consumption for required load cycles. While a forward simulation of the hydraulic drive needs an implementation of the controller which generates the control input as a function of the control error, the inverse dynamic simulation can be implemented without control. This is because the required motion is simply defined as a constraint and therefore the control error is always zero. This paper surveys examples of successful use of inverse dynamic simulation in engineering. We use the example of a hydraulic servo-drive to explain the procedure how to generate a state space description of the inverse problem from the given system of differential algebraic equations. Equation based modeling languages such as Modelica lend themselves naturally for inverse simulation because the definitions of which variables of the model are inputs and which are outputs is not made explicit in the model itself.

BACKGROUND: Cytotoxic chemotherapy/agents can cause a range of side effects in cancer patients including anemia, neutropenia and thrombocytopenia, resulting in increased morbidity and mortality. These cytopenias are in part due to massive depletion of bone marrow progenitors. A common target of many chemotherapies is the megakaryocyte (MK), a rare progenitor representing 0.2% of the bone marrow. Loss of MK lineage cells results in thrombocytopenia, therefore, discovering ways to protect MKs from cytotoxic drugs could prevent this life-threatening condition. Here, we show that eltrombopag (EP), a small molecule, non-peptide thrombopoietin receptor (TPO-R) agonist, protects healthy MKs from different cytotoxic agents. We further explored the impact of cytotoxic drugs and EP treatment on signaling molecules active in MKs. METHODS: Peripheral blood and bone marrow samples were collected from healthy donors after informed consent and following protocols in accordance with the Declaration of Helsinki. CD34+ positive cells were isolated by immuno-magnetic bead separation from the samples and expanded for 8-10 days with a cytokine cocktail to induce MK differentiation. After MK expansion, cells were transferred to 96-well plates pretreated with 14 different drugs including cytarabine, gemcitabine, paclitaxel, carboplatin, cisplatin, doxorubicin, vincristine, etoposide, midostaurin, ruxolitinib, panobinostat, azacitidine, venetoclax and navitoclax. The drugs were plated in 5 different doses in a 10,000-fold concentration range, and cells added with or without EP. After 3 days incubation, the cells were analyzed on a high throughput flow cytometer using Annexin V and 7AAD to distinguish live from dead cells. To understand the impact of the drugs and EP on signaling molecules downstream of TPO-R, we analyzed phosphorylation of AKT, ERK, STAT3 and STAT5 in populations defined by MK markers CD41a, CD42b and CD110 (TPO-R). RESULTS: We found that EP overall supports MK survival in the presence of cytotoxic agents. The greatest net effect was observed when EP was combined with BCL2 inhibitors (venetoclax, navitoclax), which resulted in increased MK cell maturation compared to inhibitor alone, while gemcitabine plus EP showed the next best response. EP combined with etoposide, vincristine, cytarabine, paclitaxel, cisplatin and ruxolitinib resulted in an increase of immature MKs without a reduction in mature MK cell numbers. Higher numbers of MKs were observed when EP was combined with midostaurin, carboplatin, panobinostat and doxorubicin, compared to treatment without EP; although these numbers were lower than other tested drugs. Phosphoflow analysis revealed intriguing differences in EP-induced signaling compared to signaling induced by recombinant human thrombopoietin (rhTPO) (Figure 1). Also, EP-induced signaling did not correlate with TPO-R expression. EP activated AKT in all MK subsets, but rhTPO induced AKT only in immature MKs. While rhTPO induced ERK phosphorylation in different MK subsets, EP had no effect on ERK activation. Furthermore, rhTPO activated STAT3 only in mature MKs, but EP induced STAT3 in all MK populations. In contrast, both EP and rhTPO induced phosphorylation of STAT5 in all MK subsets. Overall signaling was inhibited by a selected set of agents (venetoclax, etoposide, midostaurin) compared to basal signaling, but addition of EP prevented the inhibition. Similar to the cell survival analysis, venetoclax combined with EP resulted in complete recovery of signaling activity compared to venetoclax alone. But the response was not as striking for etoposide or midostaurin combination compared to treatment alone. CONCLUSION: Using MKs expanded from CD34+ cells, we found that EP supports MK survival in the presence of several different cytotoxic agents, with the most striking rescue observed when EP was combined with BCL2 inhibitors. Importantly, EP and rhTPO induced distinct signaling patterns, with EP signaling independent of TPO-R expression, suggesting alternate EP targets. The tested drugs inhibited basal signaling in MKs, but this inhibition was prevented by EP. These results suggest addition of EP to cancer treatment regimens may prevent the inhibitory effects of some agents on MKs and support future investigations to demonstrate clinical efficacy of EP in combinations with some of these cytotoxic agents to alleviate thrombocytopenia. Figure 1. Figure 1. Disclosures Javarappa: Novartis Pharma AG: Research Funding. Tsallos:Novartis: Research Funding. Marques Ramos:Novartis: Employment. Pallaud:Novartis: Employment. Heckman:Celgene: Research Funding; Novartis: Research Funding; Orion Pharma: Research Funding.