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Recently, we and others identified autophagy induction as a protective mechanism in FLT3-ITD mutant acute myeloid leukemia (AML) cells upon treatment with FLT3-inhibitors (FLT3i), constituting an increasing issue in clinical practice. Here, we investigated the molecular consequences of autophagy induction upon FLT3i treatment, exploring resistance mechanisms and interrogated whether autophagy inhibition can improve FLT3i antileukemic therapy. Drug response of primary FLT3-ITD AML samples ex vivotreated with FLT3i, quizartinib (AC220, n= 59) and midostaurin (PKC412, n=64), was associated with their transcriptional profile to unravel which molecular programs were enriched in poor responders. Both gene set enrichment analysis (GSEA) and single sample GSEA (ssGSEA) revealed enriched molecular signatures compatible with autophagy activation in poor responders to FLT3i, while sensitive samples enriched molecular signatures consonant with mitochondrial function. Enrichment scores for programs associated with “regulation of autophagy” and “autophagosome organization” were positively correlated to ex vivoresponse area under the curve values for AC220 and PKC412. To functionally address the molecular pathways associated with autophagy activation upon FLT3i treatment, MOLM-13 and MV4-11 AML cells ( FLT3-ITD) and primary AML samples, were treated with AC220 and PKC412 alone or in combination with the autophagy inhibitor chloroquine (CQ, 5-10μM). In FLT3-ITD AML models, treatment with FLT3i significantly induced the formation of acidic vesicular organelles, suggesting autophagy induction. We also observed a reduction of autophagy regulators in a dose- and time-dependent manner, evidenced by the conversion of LC3B-I to LC3B-II, with degradation of LC3B-II and p62. Furthermore, FLT3i treatment resulted in phosphorylation inhibition of AKT Ser473, mTOR Ser2448and its downstream protein p70S6K Thr421/Ser424, and phosphorylation reduction of ULK1 Ser757. Combination of FLT3i with CQ (10 μM) significantly reduced cell viability and survival for both PKC412 and AC220 inhibitors. To rule out the off target effects associated with CQ treatment, we performed a knockdown (KD) on the key autophagy mediator ATG5 using shRNA-mediated inhibition in FLT3-ITD cells. In MOLM13 cells, FLT3i treatment in ATG5-KD cells significantly reduced the IC50 (control: 31nM vs. ATG5-KD: 17nM) and increased apoptosis induction (control: 41% vs. ATG5-KD: 58%). We validated these observations in ex vivotreated primary AML samples, where we observed that in FLT3-ITD but not in FLT3wild-type samples, the combination of FLT3i (PKC412 and AC220) plus autophagy inhibitors (CQ) reduced cell proliferation and survival. In summary, we demonstrated that autophagy induction is a biological process intrinsically associated with drug resistance in FLT3-ITD mutant AMLs when treated with FLT3i. Hence, autophagy inhibition can be leveraged to increase the antileukemic effect of FLT3i and highlights the autophagy as a major acquired biological process associated with FLT3i therapy.

Natural killer (NK) cells are lymphocytes that play a critical role in both innate and adaptive immune responses. It has been shown that CD27 and CD11b are antigens expressed in the last stage of NK cells development and allow the classification of NK cells into three functional subsets. Tolerant NK cells are double negative (DN) for CD27 and CD11b, have an immature phenotype and present low secretory and cytotoxic potential. The secretory NK cells are CD27 positive, have a great ability to release cytokines, but do not exhibit cytotoxicity properties. The cytotoxic NK cells express CD11b, release granzymes and perforins, but are less capable of secreting cytokines. While cytotoxic NK cells are essential for the natural defenses against infected or malignant cells, secretory NK cells, when activated, produce different cytokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10 and others, which may modify the functions or other hematopoietic cells including the hematopoietic stem cells (HSC). We hypothesized that deregulation of the balance between secretory or cytotoxic subtypes of NK cells may favor HSC malignant transformation and contribute to stem cell-derived diseases. In this regard, few studies have shown that NK cells are defective in chronic myeloid leukemia (CML). In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. We aimed to study the activity of NK cells and their subsets in murine primary cells from MPN. NK cells subsets were quantified by flow cytometry by the use of spleen cells from a pre-established conditional vavCre knockin Jak2V617F(Jak2VF) murine model. Besides genotyping to identify controls (Jak2 wt/wt vavCre+, or Jak2WT) and Jak2-mutated mice (Jak2 wt/V617FvavCre+, Jak2VF), the presence of polycythemia and splenomegaly was confirmed in the animals with MPN. Mice (n=5 per group) were euthanized between 8-12 weeks of age. For immunophenotyping, spleen cells were isolated, submitted to red cell lyse, and stained with fluorescence-conjugated antibodies against Ter119, CD19, CD4, CD8, CD3, NK1.1 and CD27 and CD11b. All the stainings were performed at 4°C for 20 minutes and acquisition was performed in a FACSCanto™II flow cytometer (BD Biosciences). Data was analyzed by the use of the Flowjo software version X. When compared to Jak2WT controls, the Jak2VF mice had lower numbers of NK cells (1,447 x 106versus 0,381 x 106p<0.05) and T cells (0,638 x 106versus 0,158 x 106of the spleen; p<0.05) among the total splenocytes. The analysis of NK subsets showed an increase in the percentages of DN NK cells (CD27-CD11b-) in the spleen of Jak2VF mice as compared to controls (14.84% versus 4.97%; p<0.05). CD27+ immature and mature secretory NK (CD27+CD11b-/+) cells also seemed to be increased in Jak2-mutated mice but this data did not reach statistical significance (37.43% versus 17.65%; p=0.1). On the other hand, when compared to Jak2WT controls, the Jak2VF mice presented lower percentages of CD11b+ cytotoxic NK cells (77.07% versus 47.80% of the spleen cells; p<0.05). In agreement, when the expression of NK receptors was analyzed by flow cytometry, the inhibitory NKG2A receptor was found to be higher expressed in Jak2VF NK cells as compared to non-mutated cells, while the expression of the activatory receptors Ly49H and Ly49D was reduced in Jak2-mutated NK cells. Of note, low levels of the Jak2V617Fmutation were detected in sorted NK cells isolated from Jak2VF mice as compared to controls by Real Time quantitative RT-PCR, thus suggesting that the distribution between NK cell subtypes observed here is associated with the JAK2 mutational status. In summary, when compared to controls, Jak2-mutated mice presented an increased proportion of tolerant NK cells, previously described as immature cells with low secretory and cytotolytic potential. To date, CD11b-/CD27-tolerant cells have been observed among solid tumor-infiltrating NK cells, but have not been previously associated with MPN. Moreover, an inverted ratio between the secretory and cytotoxic NK subsets was also detected in Jak2VF animals, suggesting that a cytotoxic deficiency occurs and may contribute to the leukemic stem cell expansion. The expansion of the NK secretory potential deserves further elucidation. Our study provided new insights into the distribution of JAK2V617FNK functional subsets that may be used as future therapeutic targets for MPN.

Background: Despite the growing knowledge about acute myeloid leukemia (AML) pathophysiology, it remains a disease with high mortality rates, and the early recognition of patients at higher risk allows adjusted risk- or target-specific therapies. In AML, autocrine insulin like growth factor type 1 (IGF1) and its receptor (IGF1R) activation has been previously described. Free IGF mediate cellular functions through binding to the two IGF receptors, IGF1R and IGF2R. IGF2R binds to and induces degradation of IGF2, preventing IGF2-IGF1R signaling activation, IGF1R activation induces cell proliferation and prevents cell death. Therefore, IGF2R may act as a tumor suppressor and IGF1R has opposite effects. IGF1R pharmacological inhibitors have recently been investigated in the clinical setting for solid tumors, but still underexplored in hematological neoplasms. Aims: To investigate IGF1Rand IGF2RmRNA expression in primary samples from AML patients and their impact in clinical outcomes, and to analyze the effects of the IGF1R pharmacological inhibitor linsitinib in AML cell lines. Material and methods: IGF1Rand IGF2RmRNA expression levels in AML patients (n=172) were obtained from TCGA AML study (2013) available online on CBioPortal for Cancer Genomics (Cerami et al. 2012; Gao et al. 2013). Gene expression was investigated, by qPCR, in a subset AML cohort (n=101; APL=48, CBF-leukemia=53 [t(8;21)=30, Inv16=23]), and in total bone marrow samples from healthy donors (HD) (n=23). The study was approved by the ethics committee of the Institution. NB4, NB4-R2 and Kasumi-1 cell lines were submitted to linsitinib (0.25, 0.5, 1.0, 2.5, 5.0, 10, 20 or 40 µM) for 72 hours and were evaluated for cell viability (MTT assay), apoptosis (Annexin V/PI), autophagy (acridine orange staining), and protein expression/activation (western blot). Statistical analyses were performed using Student's t-test, Mann-Whitney test, Kruskal-wallis test and Spearman correlation test, as appropriate. For survival analysis, Kaplan-Meyer curves were compared with log-rank test and cox regression analyzes were applied. Results:Patients were dichotomized according to the median IGF1Rand IGF2Rexpression values, and the IGF1R/ IGF2Rratio was calculated by division of absolute number of reads coming from RNA-seq data. Patients with high IGF1R/ IGF2Rshowed poorer overall survival (OS) (29% vs.56%, P<0.01), and disease free survival (DFS) (31% vs.45%, P=0.015). Individually, high IGF1R(33% vs.51%; P=0.042) and low IGF2R(32% vs.52%; P=0.012) were associated with a shorter OS. By Cox regression analysis, IGF1R/ IGF2R(P=0.039), cytogenetic risk stratification (P<0.001), age (P<0.001) and white blood cell counts (P=0.003) independently predicted poorer OS. AML patients harboring t(15;17) or t(8;21) presented higher levels of IGF1R/ IGF2Rwhen compared with other AML patients (P≤0.01). Using a subset of APL and CBF-leukemia, a higher IGF1R/ IGF2Rratio was observed in AML patients with t(15;17) or t(8;21) compared with healthy donors (HD) (P<0.01). IGF1Rand IGF2Rexpression positively correlated in HD (r=0.9296; P<0.001), and negatively correlated in AML TCGA study (r=-0.2366; P=0.0018). In NB4, NB4-R2 and Kasumi-1 cells, linsitinib ≥1 µM reduced cell viability (P<0.05) and significantly increased apoptosis, as demonstrated by increased cleaved caspase 3 and annexin V+ cells. The mean percentage of annexin V+ cells for control, linsitinib 10, 20 and 40 µM were 3,9%, 14%, 32% and 93% for NB4, 6%, 24%, 53% and 96% for NB4-R2 and 7%, 19%, 49% and 96% for Kasumi-1 (all P<0.05), respectively. Linsitinib 10 µM increased autophagy in at least 3 fold-change in all cell lines tested (all P<0.05). Linsitinib 10 µM plus the autophagy inhibitor chloroquine 20 µM increased apoptosis when compared with monotherapy in NB4 cells (45% vs15%), NB4-R2 cells (68% vs15%) and Kasumi-1 (35% vs15%) (all P<0.05). The IGF1R pharmacological inhibitor reduced phosphorylation/activation of IGF1R (Tyr1135), IRS1/2 (Tyr612), AKT1/2/3 (Ser473), P70S6K (Thr421/Ser424), 4EBP1 (Thr70) and ERK1/2 (Thr185/Tyr187). Conclusions:In AML,increased IGF1R/ IGF2Rpredicts poorer survival outcomes and the pharmacological IGF1R inhibitor linsitinib exerts an anticancer activity and downregulates PI3K/AKT and MAPK signaling. In the concept of multi target treatment, IGF1R arise as a promising therapeutic target for patients with AML.

Introduction:Chronic myeloid leukemia (CML) is a hematological malignancy associated with the BCR-ABL1 oncoprotein, which results in deregulated kinase activity-driven leukemic phenotype. Most CML cases are successfully treated with tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1, but a significant percentage of patients develop drug resistance and/or progress to blast crisis, a disease stage that is refractory to therapy. Furthermore, recent studies have provided evidence that the CML leukemia stem cell is resistant to imatinib treatment. In this sense, the identification of other crucial proteins that cooperate with BCR-ABL1 signaling pathways may represent secondary therapeutic targets optimize treatment strategies. Increased IRS1mRNA expression has been previously identified as positively correlated with worse overall survival in BCR-ABL1-positive adult acute lymphoblastic leukemia. In K562 cells, IRS1 has been identified as a binding partner of BCR-ABL1 and is capable of activating the PI3K/Akt/mTOR and MAPK pathways. Recently, a pharmacological IRS1/2 inhibitor (NT157) has been developed and has shown promising results in preclinical studies on solid tumors. We have previously shown that NT157 treatment reduces colony formation of primary CML cells but not in normal cells, decreases cell viability and proliferation of K562 cells, and increases apoptosis of K562 cells in a time- and dose-dependent manner (ASH 2015 - Abstract #83876). To further characterize the mechanism of the sensitivity of CML cells to IRS1/2 inhibition, here we performed PCR-based gene expression analyses in CML cells in response to NT157 treatment and investigated the effects of IRS1/2 inhibition on cell proliferation and apoptosis in Ba/F3 cells expressing wild-type or T315I mutant BCR-ABL1.

Background:Acute lymphoblastic leukemia (ALL) is an aggressive cancer of immature progenitors that shows aberrant activation of signaling pathways. The IGF1/IGF1R signaling pathway is initiated through binding of the ligand (IGF1) to its transmembrane receptor (IGF1R), and subsequent activation of IRS1 and IRS2 proteins activating PI3K/Akt/mTOR and MAPK pathways, which are important signaling pathways reported to contributes to pathogenesis of ALL. However, the therapeutic potential of IGF1R/IRS signaling has not been previously studied in ALL cells.