Search

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.

In myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN), bone marrow (BM) histopathology is assessed to identify dysplastic cellular morphology, cellularity, and blast excess. Yet, other morphologic findings may elude the human eye. We used convolutional neural networks to extract morphologic features from 236 MDS, 87 MDS/MPN, and 11 control BM biopsies. These features predicted genetic and cytogenetic aberrations, prognosis, age, and gender in multivariate regression models. Highest prediction accuracy was found for TET2 [area under the receiver operating curve (AUROC) = 0.94] and spliceosome mutations (0.89) and chromosome 7 monosomy (0.89). Mutation prediction probability correlated with variant allele frequency and number of affected genes per pathway, demonstrating the algorithms' ability to identify relevant morphologic patterns. By converting regression models to texture and cellular composition, we reproduced the classical del(5q) MDS morphology consisting of hypolobulated megakaryocytes. In summary, this study highlights the potential of linking deep BM histopathology with genetics and clinical variables. Significance: Histopathology is elementary in the diagnostics of patients with MDS, but its high-dimensional data are underused. By elucidating the association of morphologic features with clinical variables and molecular genetics, this study highlights the vast potential of convolutional neural networks in understanding MDS pathology and how genetics is reflected in BM morphology. See related commentary by Elemento, p. 195.

The immunologic microenvironment in various solid tumors is aberrant and correlates with clinical survival. Here, we present a comprehensive analysis of the immune environment of acute myeloid leukemia (AML) bone marrow (BM) at diagnosis. We compared the immunologic landscape of formalin-fixed paraffin-embedded BM trephine samples from AML (n = 69), chronic myeloid leukemia (CML; n = 56), and B-cell acute lymphoblastic leukemia (B-ALL) patients (n = 52) at diagnosis to controls (n = 12) with 30 immunophenotype markers using multiplex immunohistochemistry and computerized image analysis. We identified distinct immunologic profiles specific for leukemia subtypes and controls enabling accurate classification of AML (area under the curve [AUC] = 1.0), CML (AUC = 0.99), B-ALL (AUC = 0.96), and control subjects (AUC = 1.0). Interestingly, 2 major immunologic AML clusters differing in age, T-cell receptor clonality, and survival were discovered. A low proportion of regulatory T cells and pSTAT1+cMAF− monocytes were identified as novel biomarkers of superior event-free survival in intensively treated AML patients. Moreover, we demonstrated that AML BM and peripheral blood samples are dissimilar in terms of immune cell phenotypes. To conclude, our study shows that the immunologic landscape considerably varies by leukemia subtype suggesting disease-specific immunoregulation. Furthermore, the association of the AML immune microenvironment with clinical parameters suggests a rationale for including immunologic parameters to improve disease classification or even patient risk stratification.

Background: Measurable Residual Disease (MRD) assessments are gaining increasing acceptance as a prognostic factor for tailoring treatment in hematological malignancies. Acute Myeloid Leukemia (AML) is a heterogeneous disease with high relapse rates and presents a high unmet need for effective treatment options. Measurement of residual disease after therapy reflects a combination of all resistance mechanisms and is currently used for guiding treatment options. Study Design: In this study, we aimed to validate an AML-MRD assay by multiparameter flow cytometry (MFC) methodology. This is a 4-tube, 8-parameter assay designed to incorporate cell differentiation (CD) markers for identification of a diverse group (covering roughly 90% of patients, Cloos et al, 2018) of Leukemia Associated Immunophenotypes (LAIPs) to accurately identify both native phenotypes and phenotype shifts after drug treatment. These CD markers were selected based on extensive investigation of many markers and in line with the consensus recommendations from European Leukemia Network AML working party (Schuurhuis et al, 2018), while specimen testing and interpretation principles were performed in accordance with Cloos et al, 2018. The assay validation focused on evaluation of sensitivity (MRD cut point and LOD), precision and accuracy as key criteria for evaluating assay performance utilizing primary patient specimens and AML cell lines representing different LAIPs. The results were orthogonally verified in a blinded manner by morphologic assessment at Navigate and by the MRD-team at VUMC Amsterdam. Results: Two experimental approaches were adopted to evaluate analytical and functional sensitivity (clinical applicability) of the assay. Results indicated analytical sensitivity (LOD) as low as 0.01% LAIPs of total WBC and functional sensitivity (LOQ) of 0.1% (MRD cut point). Excellent repeatability and reproducibility (less than 20% CV) was observed across instruments, operators and independent measurements (n = 75). The frequencies of AML blasts detected by MFC and morphological examination were highly concordant (Spearman r = 0.95, P value < 0.001, n = 24). LAIPs deduced across nine patient specimens by the Navigate laboratory were independently confirmed by the MRD-team at VUMC Amsterdam. Conclusion: In summary, based on the use of consensus markers recommended by ELN for reliable capture of a broad group of LAIPs in AML patients and verification of key assay performance characteristics, we believe this comprehensive MFC based AML MRD assay is fit-for-purpose for accurately assessing measurable residual disease. Following clinical trial validation, MRD might be used as a surrogate endpoint for approval of emerging agents. Disclosures Marques Ramos: Novartis: Current Employment. Larson:BMS, Bioline, Celgene, Juno, Janssen: Research Funding; TORL Biotherapeutics: Current equity holder in private company. Sarikonda:Novartis: Current Employment.

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.

Azacitidine treatment of myelodysplastic syndromes (MDSs) generally exacerbates thrombocytopenia during the first treatment cycles. A Study of Eltrombopag in Myelodysplastic Syndromes Receiving Azacitidine (SUPPORT), a phase 3, randomized, double-blind, placebo-controlled study, investigated the platelet supportive effects of eltrombopag given concomitantly with azacitidine. International Prognostic Scoring System intermediate-1, intermediate-2, or high-risk MDS patients with baseline platelets

Eltrombopag (EP), a small molecule thrombopoietin receptor (TPO-R) agonist and potent intracellular iron chelator, has shown remarkable efficacy in stimulating sustained multilineage hematopoiesis in patients with bone marrow failure syndromes, suggesting an effect at the most immature hematopoietic stem and multipotent progenitor level. Although the functional and molecular effects of EP on megakaryopoiesis have been studied in the past, mechanistic insights into its effects on the earliest stages of hematopoiesis have been limited. Here, we investigated the effects of EP treatment on hematopoietic stem cell (HSC) function using purified primary HSCs in separation-of-function mouse models, including a TPO receptor-deficient strain, and stem cells isolated from patients undergoing TPO-R agonist treatment. Our mechanistic studies showed a stimulatory effect on stem cell self-renewal independently of TPO-R. Human and mouse HSCs responded to acute EP treatment with metabolic and gene expression alterations consistent with a reduction of intracellular labile iron pools that are essential for stem cell maintenance. Iron preloading prevented the stem cell-stimulatory effects of EP. Moreover, comparative analysis of stem cells in the bone marrow of patients receiving EP showed a marked increase in the number of functional stem cells compared to patients undergoing therapy with romiplostim, another TPO-R agonist lacking an iron-chelating ability. Taken altogether, our study demonstrates that EP stimulates hematopoiesis at the stem cell level through iron chelation-mediated molecular reprograming and indicates that labile iron pool-regulated pathways can modulate hematopoietic stem cell function.

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 Following the positive outcome of the RATIFY phase 3 clinical trial, the multi-kinase inhibitor midostaurin was approved for the treatment of adult patients with newly diagnosed FLT3-mutated acute myeloid leukemia (AML). However, we and others have observed that single agent midostaurin yields responses also in a substantial portion of patients not carrying FLT3 mutations. The molecular basis and the kinase targets mediating these responses are poorly understood and no biomarkers predictive of response in FLT3 wildtype (wt) AML patients exist. To identify markers distinguishing the FLT3 wt responding subset of patients, we trained machine learning multi-marker models using AML patient baseline transcriptomic and mutational data to predict ex vivo responders vs. non-responders. Further, to better understand the molecular basis of midostaurin responses and to explore the unique signaling networks modulated by midostaurin, we profiled the sensitivities of AML patient samples to midostaurin in comparison to, and in combination with, several clinically relevant oncological targeted agents of diverse mechanistic classes. Results Midostaurin target space is unique and it retains anti-leukemic potency under cytoprotective conditions. We have previously established that single agent midostaurin is effective ex vivo in about 25% of FLT3 wt AML patient samples and retains potency in a cytoprotective medium that masks the effects of more selective FLT3 inhibitors such as quizartinib, crenolanib and sorafenib (Karjalainen et al, Blood 2017). To further investigate the unique pathways that midostaurin, but not other FLT3 inhibitors targets, we correlated the response patterns of 87 AML patient samples in cytoprotective medium to midostaurin and 261 other kinase inhibitors in our oncology compound collection. In unsupervised cluster analysis, midostaurin showed highly similar response patterns to AZD7762, OTS167, milciclib, pacritinib, ENMD-2076 and fostamatinib. Publicly available in vitro kinase profiling (Tang et al, Cell Chem. Biol. 2018) suggested that midostaurin does not inhibit most of the primary targets of these other inhibitors, with only aurora kinases, JAK kinases and SYK appearing to be shared potent targets. Midostaurin anti-leukemic potency is determined by the mutational background. Several multi-marker, supervised machine learning models were compared to extract biomarker signatures from either baseline transcriptomic or mutational data, in the task of predicting ex vivo midostaurin response in samples cultured in cytoprotective medium. In the full cohort (N=81), the presence of FLT3 mutations (both internal tandem repeat and tyrosine kinase domain mutations) was the strongest predictor of response. In the FLT3 wt cases (N=49), our results revealed that other select mutations correlated well with either response or non-response upon Bayesian Linear Regression analysis with cross-validation (Ammad-Ud-Din et al, Bioinformatics, 2017). Mutations in PTPN11, U2AF1, SRSF2, RUNX1, JAK2 and BCOR predicted midostaurin responders, while mutations in GATA2, WT1, NPM1 and IDH2 were enriched in non-responders (Figure 1). Baseline transcriptomic profiles, however, did not provide added value for the predictive power. Midostaurin efficacy can be enhanced by combination with other targeted agents. Combinatorial drug screening of midostaurin in cytoprotective medium revealed several synergizing drug classes, including BCL-2 and MDM2-p53 inhibitors. Further analysis of synergizing agents in broader AML patient sample cohorts is ongoing. Conclusions Our results show that midostaurin may reach its biological effects through inhibition of additional kinases than just FLT3. In both FLT3 mutant and wt cases, midostaurin responses are influenced by the overall mutational background. Furthermore, our data indicates that midostaurin efficacy can be enhanced through combination with other agents. Together, we have significantly expanded the understanding of molecular determinants of midostaurin response in primary AML cells, supporting predictive biomarker discovery efforts and development of synergistic drug combinations. The emerging hypotheses from this work will have to be tested in clinical studies. Disclosures Porkka: Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Marques Ramos:Novartis: Employment. Pallaud:Novartis: Employment. Aittokallio:Novartis: Research Funding. Wennerberg:Novartis: Research Funding.

β-Thalassemia is one of the most common genetic red blood cell (RBC) disorders characterized by reduced (such as in non-transfusion dependent thalassemia or NTDT) or absent (such as in transfusion dependent thalassemia or TDT) production of β-globin chains. Ineffective erythropoiesis (IE) with consequent anemia leads to extra-medullary hematopoiesis (EMH), splenomegaly and iron overload mediated by low levels of hepcidin. We previously demonstrated that IE in β-thalassemia is associated with increased proliferation and reduced differentiation of erythroid progenitors (Libani et al, 2008, Blood). This is mediated by increased production of erythropoietin (EPO), which activates the downstream JAK2 kinase in erythroid progenitors. As a consequence, hepatosplenomegaly may result, often requiring splenectomy to prevent serious morbidities and mortality. The increased synthesis of EPO in thalassemia is the result of hypoxia. Hypoxia Inducible Factor-2a (HIF2a) is a central mediator of cellular adaptation to hypoxia and stimulates renal and hepatic EPO synthesis. Furthermore, splenomegaly in thalassemia also exacerbates the anemia, as a large proportion of the circulating RBC are engulfed and eliminated by an enlarged spleen. Therefore, we hypothesized that targeting the EPO/JAK2 pathway limits the number of erythroid progenitor cells and reduces the splenomegaly, and it could be utilized as an alternative to splenectomy. We utilized JAK2 inhibitors or HIF2a antisense oligonucleotides (HIF2a-ASO) to target the EPO/JAK2 pathway. Moreover, we hypothesized that combination of these drugs with blood transfusion therapy will further reduce the splenomegaly and, eventually, improve the blood transfusion regimen. We tested two commercially available JAK2 inhibitors in mice affected by NTDT (Hbbth3/+): INCB018424 (Ruxolitinib) and TG101348 (Fedratinib, SAR302503). Both drugs were administered for 10 days, twice daily by oral gavage at a dose of 180 and 120 mg/Kg respectively. A mild reduction in hemoglobin (Hb) levels, (in the range of 9%), was observed in animals treated with both inhibitors when compared to vehicle- treated mice. Splenomegaly was significantly reduced with both inhibitors (up to 56% in reduction). Flow cytometry studies on spleen cells revealed that animals receiving the inhibitors exhibited a reduction in the number of erythroid progenitors compared to the placebo-treated animals . In parallel, we performed pharmacokinetic[BM1] studies using Hif2a-ASO. Animals received Hif2a-ASO at a dose of 25 mg/kg twice weekly for 10 days or 3 weeks by IP injections. After 10 days of treatment the spleen weight was reduced 58%, while the Hb level was reduced in the range of 27%%. After 3 weeks the effect observed on the anemia was more pronounced, with a reduction of more that 40% in Hb levels, while the spleen reduction was 36%. We then compared and combined these drugs with blood transfusion using Hbbth3/+ mice. Blood transfusion reduced splenomegaly 49% and 53% when compared, respectively, to non-transfused controls and animals treated with JAK2 inhibitors alone. When transfusion was combined with the administration of JAK2 inhibitors for 10 days, the spleen size was further reduced (72% when compared with non-transfused controls). Combination of Hif2a-ASO and blood transfusion is in progress. We then tested the JAK2 inhibitors in mice affected by TDT. TDT animals are generated by engrafting WT mice with Hbbth3/th3 fetal liver cells. Following engraftment, these mice show a large splenomegaly and rapidly become dependent on chronic blood transfusion for survival (Gardenghi et al, 2007, Blood). Preliminary studies suggest that administration of JAK2 inhibitors for 10 days, together with blood transfusion, further reduces spleen weight by 71% compared to transfusion alone. Combination of Hif2a-ASO and blood transfusion in these animals is also in progress. In summary, JAK2 inhibitors and Hif2a-ASO reduce splenomegaly by targeting the EPO/JAK2 pathway and limiting the excessive proliferation of erythroid cells. Therefore, these drugs could be effective in reversing the splenomegaly and may offer an important approach to splenectomy. Additional studies are in progress to evaluate the outcome of combination therapy on the efficacy of transfusions. [BM1]These aren't pharmacokinetic studies. Suggest just striking the word Disclosures Casu: Medgenics LLC: Research Funding; Ionis Pharmaceuticals: Research Funding; Merganser Biotech: Research Funding. Aghajan:Ionis Pharmaceuticals: Employment, Equity Ownership. Guo:Ionis Pharmaceuticals: Employment, Equity Ownership. Melchiori:Adaptimmune Ltd: Employment. Ramos:Novartis: Employment.

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.

Background The PI3K pathway is hyperactivated in many cancers, including 70 % of breast cancers. Pan- and isoform-specific inhibitors of the PI3K pathway are currently being evaluated in clinical trials. However, the clinical responses to PI3K inhibitors when used as single agents are not as efficient as expected. Methods In order to anticipate potential molecular mechanisms of resistance to the p110α isoform-selective inhibitor BYL719, we developed resistant breast cancer cell lines, assessed the concomitant changes in cellular signaling pathways using unbiased phosphotyrosine proteomics and characterized the mechanism of resistance using pharmacological inhibitors. Results We found an increase in IGF1R, IRS1/IRS2 and p85 phosphorylation in the resistant lines. Co-immunoprecipitation experiments identified an IGF1R/IRS/p85/p110β complex that causes the activation of AKT/mTOR/S6K and stifles the effects of BYL719. Pharmacological inhibition of members of this complex reduced mTOR/S6K activation and restored sensitivity to BYL719. Conclusion Our study demonstrates that the IGF1R/p110β/AKT/mTOR axis confers resistance to BYL719 in PIK3CA mutant breast cancers. This provides a rationale for the combined targeting of p110α with IGF1R or p110β in patients with breast tumors harboring PIK3CA mutations. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0697-1) contains supplementary material, which is available to authorized users.