ALBERT

Beschreibung: Acute myeloid leukemia (AML) stem cells (LSC) are an extremely rare fraction of the overall disease (likely

Beschreibung: Approximately 30% of patients with acute myeloid leukemia (AML) harbor activating mutations in the fms-like tyrosine kinase receptor 3 (FLT3). Such mutations are associated with increased propensity to relapse and dramatically decreased survival. FLT3 tyrosine kinase inhibitors (TKI), such as quizartinib, have shown modest clinical effects as single agents, and resistance remains a significant clinical problem. In addition to constitutive activity of the FLT3 receptor, AML cells with an internal tandem duplication of FLT3 (FLT3-ITD) exhibit elevated levels of reactive oxygen species (ROS). Antioxidants such as heme-oxygenase 1 (HO-1) are often upregulated in conjunction with increased ROS levels. In addition to its role as an antioxidant, HO-1 also has known proliferative and anti-apoptotic functions in some cell types. A study in non-small cell lung carcinoma recently suggested that HO-1 inhibits the Notch pathway through binding the Notch receptor. As Notch signaling exerts pro-apoptotic effects in AML, targeting HO-1 may induce Notch signaling, representing a potential therapeutic approach. Our data show that HO-1 is particularly elevated in FLT3-ITD expressing AML cells. Interestingly, HO-1 protein was found to be further elevated in AML cells with acquired resistance to FLT3-directed TKI compared to TKI-sensitive cells. Importantly, knockdown of HO-1, or inhibition of HO-1 catalytic activity with zinc protoporphyrin (ZnPP), leads to decreased survival and proliferation, demonstrating a critical pro-survival role of HO-1. We have determined that expression of HO-1 is under the control of NRF2, a transcriptional regulator of the antioxidant response, in FLT3-ITD positive AML cells. Inhibition of NRF2 with brusatol resulted in increased sensitivity to quizartinib in TKI-sensitive cell lines. Further, brusatol restored sensitivity to low nanomolar concentrations of quizartinib in TKI-resistant cells. Together, our work shows that combined inhibition of FLT3 and NRF2 using quizartinib and brusatol, respectively, exhibits synergy in the context of FLT3-ITD AML. Given the reported interaction between HO-1 and Notch, we sought to determine whether this interaction occurs in AML. Using the TCGA AML database, we found significant co-occurrence of HMOX1 (HO-1) and NOTCH2 mRNA expression in 200 patient samples. In vitro immunoprecipitation of the Notch2 receptor revealed that HO-1 directly interacts with Notch2 in FLT3-ITD positive AML cells. Importantly, brusatol treatment resulted in activation of the Notch pathway as demonstrated by cleavage of the Notch receptor and increased transcript levels of HES1, a canonical downstream target of Notch signaling. Given that HO-1 interacts with Notch2 and is under NRF2 control, we propose that decreasing HO-1 levels with brusatol treatment will modulate the Notch pathway such that its anti-leukemic effects can be exerted. In conclusion, inhibition of HO-1, directly or through NRF2 inhibition, demonstrates synergistic effects with FLT3 inhibition in both TKI-sensitive and -resistant AML. Furthermore, NRF2 inhibition induces Notch activation, providing a potential mechanism for this synergistic combination. Our data suggest that antioxidant modulation is a potential therapeutic approach for FLT3-ITD positive AML. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Acute myeloid leukemia (AML) is a conglomerate of hematologic malignancies characterized by recurrent genetic and/or chromosomal aberrations. Recent development of targeted agents has bolstered our armamentarium of therapeutic options and improved outcomes for many patients. Acquiring deeper mechanistic understanding of myeloid leukemogenesis will provide a basis for development of even more therapeutic strategies and further improve patient outcomes. Our clinical data have revealed that overexpression of HNRNPK is a recurrent abnormality that occurs in upwards of 20% of AML cases at both the RNA and protein levels. Using bone marrow samples from a similar proportion of patients, we have recently discovered a supernumerary marker chromosome containing an extra copy of the HNRNPK locus that is not detectable with routine cytogenetic testing. We have further associated high hnRNP K protein levels with decreased overall survival in de novo AML, emphasizing the need to understand the role of hnRNP K in myeloid malignancy. To directly evaluate the oncogenic capacity of hnRNP K, we have overexpressed hnRNP K in murine fetal liver cells (FLCs). Using CyTOF and colony formation assays, we demonstrated that hnRNP K-overexpressing FLCs have altered differentiation potential and self-renewal capacity compared to empty vector controls in vitro. These findings are recapitulated in vivo, as murine recipients of hnRNP K-overexpressing FLCs develop myeloid lineage disease, often manifesting as fatal megakaryocytic leukemia. To elucidate a mechanism by which hnRNP K causes myeloid disease, we performed hnRNP K immunoprecipitation followed by mass spectrometry in an AML cell line and identified that hnRNP K preferentially interacts with translational machinery, ribosomal subunits, and proteins involved in RNA processing. In conjunction with data from our hnRNP K overexpression models that indicate overexpression of hnRNP K occurs primarily in the cytoplasm, we then performed hnRNP K-RNA immunoprecipitation followed by sequencing (RIP-Seq). We determined that hnRNP K interacts with the transcript of RUNX1-a master regulator of hematopoiesis and a critical player in a myriad of leukemias-including megakaryocytic leukemias like those observed in our mouse models. Using biochemical assays, we have demonstrated that hnRNP K directly binds to consensus sequences in the RUNX1 transcript, and ultimately alters RUNX1 translation. Indeed, mice exhibiting hnRNP K overexpression have increased protein levels of Runx1 in hematopoietic tissues. Our data demonstrate that hnRNP K overexpression drives myeloid malignancy. Currently, we are screening compounds that will disrupt the interaction between hnRNP K and RUNX1 in our efforts to further understand myeloid biology and ultimately improve outcomes for patients with these diseases. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Background: The mechanisms of resistance to immunotherapies in patients with acute myeloid leukemia (AML) are not well characterized and biomarkers for improved immunotherapeutic strategies are critical. Multiple phase II/III clinical trials combining hypomethylating agents (HMA), azacitidine (AZA) and decitabine, with immune checkpoint inhibitors are now underway for patients with newly diagnosed and relapsed/refractory (R/R) AML and MDS based on data suggesting that 1) hypomethylating agents increase tumor expression of PD-L1 and PD-1 in myeloid malignancies(Yang H et al. Leukemia 2014); 2) blocking the PD-1/PD-L1 signaling axis has profound anti-leukemic response (Zhou et al. Blood 2011); and 3) clinical responses to Azacitidine/PD1 inhibitor (nivolumab) in relapsed AML (Daver et al., Cancer Discovery). Here, we characterize the baseline immune landscape and potential mechanisms of resistance using single-cell mass cytometry (CyTOF) profiling of serially collected samples from R/R AML patients undergoing therapy with AZA and PD-L1 inhibitor avelumab [NCT02953561]. Methods: Bone marrow (BM) and peripheral blood (PB) samples were collected from 9 patients prior to treatment with AZA and avelumab and after cycles 1, 3, and 5 (as available). To interrogate immune profiling in these samples, we have developed and optimized a novel CyTOF antibody panel that includes immunophenotypic markers to distinguish AML stem cells and blasts from adjacent immune cell subsets (T, B, NK cells, and monocytes), as well as known checkpoint ligands and receptors. Data was analyzed using the uniform manifold approximation and projection (UMAP) algorithm (McInnes et al. arXiv 2018) implemented through Cytofkit (Chen et al. PLoS Comput Biol 2016). Results: We first used the multi-parameter immunophenotyping to characterize the immune microenvironment in normal and leukemic BM prior to therapy. At baseline, the composition of AML BM CD4 and CD8 T cells contained a significantly smaller fraction of naïve T-cells when compared to healthy bone marrow controls (both p

Beschreibung: Background The long-term survival of AML, especially of older AML patients, under the established therapies remains poor. A recent breakthrough therapy of selective BCL-2 inhibitor venetoclax with hypomethylating agents (HMA) azacitidine or decitabine is approved to treat older and unfit patients with AML (DiNardo et.al. EHA 2020). This combination induces responses in 〉 65% of older AML patients, but the responses in relapsed/refractory AML patients are sub-optimal. Upregulation of programmed death-1 (PD-1) in T-cells is associated with AML immune-suppression, and combination of anti-PD-1 with azacitidine has activity in relapsed/refractory AML (Daver et. al. Cancer Discovery 2019). Recent pre-clinical findings indicate that venetoclax preserved T-cells immunity by sparing central memory T-cells and enhanced activity of anti-PD-1 antibodies in immune-competent mouse models in vivo (Mathew et.al. Blood 2018). In this study, we tested the hypothesis that PD-1 inhibition facilitates the depth and duration of response to venetoclax and HMA combination by reactivating T-cells mediated immunity against AML. Results We collected peripheral blood (PB) samples from trial patients before and after receiving the treatment of decitabine and venetoclax (Dec_Ven) (NCT03404193) and examined the percentage of AML blasts and T-cells by multi-parameter flow cytometry. The combination of Dec_Ven effectively reduced PB CD33+/CD34+cells from 46 ± 15% at baseline (BL) to 27 ± 16% at 2.3 days (Day 1 - 3) (time point 1, or T1), and 15 ± 13% at 5.5 days (Day 3 - 8) (T2), and increased CD3+ T-cells: 28 ± 10% (BL), 54 ± 13% (T1) and 59 ± 19 % (T2), (BL vs.T1, CD33/CD34+, p = 0.001; CD3+, p = 0.02, n = 6). The reduction of CD33+/CD34+ positively correlated with the depletion of circulating blasts (R = 0.64, p = 0.0001). Further analyzing the T-cells subsets among CD4helper cells and CD8cytotoxic cells, we found that Dec_Ven therapy promoted an activated T-cells phenotype by upregulating CD69 and PD-1 in both CD4and CD8cells in early time point samples, and led to T-cells exhaustion, as indicated by persistent expression of PD-1 at later time-point when CD69 was undetectable (PD-1 in CD3+CD4+ cells: 13 ± 2%, n = 6 (BL), 15 ± 3%, n = 6 (T1) and 22 ± 7%, n = 5 (T2); PD-1 in CD3+CD8+ cells: 14 ± 3%, n = 6 (BL), 27 ± 5%, n = 6, (T1), 29 ± 9%, n =5 (T2), BL vs. T1, p = 0.05). The persistent expression of PD-1 is a hallmark of T-cells exhaustion and immunosuppression (Jia et.al. Blood Cancer J 2018). Mass cytometry CyTOF immune-profiling of various compartments of CD4and CD8cells on PB uncovered that Dec_Ven reduced naïve T-cells (CD45RA+CCR7+) but spared or even increased frequencies of the central (CD45RA-CCR7+) and effector memory (CD45RA-CCR7-) CD4 or CD8 cells. In parallel, we measured the serum cytokines levels, and found that treatment of Dec_Ven increased IL2, IL6 and INFg secretion in PB. IL2 and IL6 are two cytokines that regulate the proliferation and differentiation of effector memory T-cells; and INFg is a critical mediator of treatment-induced immune response. To further test if targeting PD-1 induced anti-leukemia effect and amplified the effect of Dec_Ven, we treated leukemia cells collected before and after Dec_Ven therapy with anti-CD3 and anti-PD-1 antibodies in vitro. Treatment reduced CD33+/CD34+ cells in 2 of 3 pre-treatment samples (untreated vs. anti-CD3_PD-1: 55% vs. 15%, pt #6; 70% vs. 52%, pt #7) and synergistically reduced/eliminated the residual CD33+/CD34+ cells in AML samples obtained after Dec_Ven therapy (untreated vs. anti-CD3_PD-1: 84% vs. 66% Day 3, pt #3; 18% vs. 8% Day 3, pt #6; 44% vs. 35% Day 1, 5% vs. 2% Day 4, pt #7) (Fig.1). Conclusions Our data indicate that Dec_Ven therapy induces PD-1 expression on T-cells but selectively preserves potentially important anti-tumor T-cells immune subsets. The "triple" combination therapy of HMA with BCL-2 and PD-1 antagonists may facilitate anti-leukemia responses by enhancing T-cells function. Clinical trial to test this hypothesis is ongoing in relapsed/refractory AML patients (NCT02397720). Disclosures DiNardo: Celgene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Jazz: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Calithera: Research Funding; Novartis: Consultancy; MedImmune: Honoraria; Takeda: Honoraria; Syros: Honoraria; ImmuneOnc: Honoraria; Notable Labs: Membership on an entity's Board of Directors or advisory committees. Daver:Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Servier: Research Funding; Genentech: Research Funding; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novimmune: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Trovagene: Research Funding; Fate Therapeutics: Research Funding; ImmunoGen: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Consultancy, Membership on an entity's Board of Directors or advisory committees; Trillium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Konopleva:Agios: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Eli Lilly: Research Funding; Calithera: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Ablynx: Research Funding; AbbVie: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Genentech: Consultancy, Research Funding.

Beschreibung: The inferior cure rate of T-cell acute lymphoblastic leukemia (T-ALL) is associated with inherent drug resistance. The activating NOTCH1 gene mutations have been reported to cause chemoresistance at the stem cell level1. Direct NOTCH1 inhibition has failed in clinical trials due to a narrow therapeutic window but targeting key oncogenic and metabolic pathways downstream of mutated NOTCH1 may offer novel approaches. We previously reported that rapid transformation of thymocytes at the DN3 differentiation stage into preleukemic stem cells (pre-LSC) requires elevated Notch1 in addition to the presence of Scl/Lmo11. Notably, we showed that cellular metabolism of NOTCH1-mutated T-ALLs depends on Oxidative Phosphorylation (OxPhos) and that OxPhos inhibition using the complex I inhibitor IACS-010759 (OxPhos-i) is efficacious in NOTCH1-mutated T-ALL patient derived xenografts (PDXs)2. Here, we investigated the link between NOTCH1-mutated chemoresistance and OxPhos in pre-leukemic and leukemic cells, utilizing comprehensive molecular and functional assays. We hypothesized that chemotherapy aided by OxPhos-i overcomes chemoresistance, depletes LSCs and combats T-ALL. First, we analyzed the role of OxPhos in downstream Notch1 targets at the pre- and leukemic stage considering four stages of thymocyte differentiation (D1-D4), in a mouse model of human T-ALL1. Gene set enrichment analysis (GSEA) implicated increased expression of Notch1 target genes starting at DN1, and OxPhos target genes were the highest-ranked gene set at DN3. Next, activation of Notch1 by its ligand DL4 and inhibition of OxPhos reduced viability of pre-LSCs, indicating that ligand-dependent activation of Notch1 signaling upregulates the OxPhos pathway and sensitizes pre-LSCs to OxPhos-i. To clarify the role of Notch1 signaling, we examined the effect of IACS-010759 on pre-leukemic thymocytes harboring LMO1, SCL-LMO1, NOTCH1, LMO1-NOTCH1 and SCL-LMO1-NOTCH1 with and without DL4 stimulation. We found that in the absence of DL4, only thymocytes harboring the Notch1 oncogene responded to OxPhos-i, whereas all DL4-stimulated thymocytes responded regardless of Notch1 status (Fig. 1a). In addition, at the leukemic stage, we found elevation of the OxPhos pathway driven by oncogenic Notch1 when we compared transcriptomes of SCL-LMO1 induced T-ALL in the presence or absence of the NOTCH1 oncogene. In line with the murine T-ALL NOTCH1 model, we performed transcriptome analysis of two independent T-ALL patient cohorts prior to chemotherapy, COG TARGET ALL (n=263) and AALL1231 (n=75), comparing transcriptomes of NOTCH1-mutated vs NOTCH1-wt T-ALLs. We found co-segregation of NOTCH1 mutations with significant upregulation of OxPhos and TCA cycle genes and downregulation of apoptosis signaling. Aiming to reverse the NOTCH1-controlled anti-apoptotic program and chemoresistance, we next tested the combination of Vincristine, Dexamethasone and L-Asparaginase (VXL) with IACS-010759. When compared to vehicle, OxPhos-i or VXL alone, only the VXL-OxPhos-i treatment caused an energetic crisis indicated by decreased OCR and ECAR (Seahorse), which translated to a profound reduction of viability (CTG, flow cytometry) in T-ALL cell lines (n=9) and primary T-ALL samples (n=5). Additionally, the IACS-VXL combination in vivo resulted in pan-metabolic blockade, which caused metabolic shut-down and triggered early induction of apoptosis in leukemic cells in peripheral blood, spleen and bone marrow (Fig. 1b). Single cell Proteomic analysis (CyTOF) of spleen showed reduced expression of cell proliferation marker -ki67, c-myc, ERK and p38 proteins, and reduction in number of leukemic cells. Finally, this combination therapy resulted in reduced leukemia burden and extension of overall survival across all three aggressive NOTCH1-mutated T-ALL PDX models (p

Beschreibung: Background: Acute myeloid leukemia (AML) stem cells (LSC), the likely source of relapsed disease, are capable of surviving current standard chemotherapy. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression datasets and identified CD200 as significantly over-expressed in LSCs when compared to paired blast cells, as well as when compared to their normal hematopoietic stem cell (HSC) counterparts (Fig 1A; Herbrich et al Blood. 2018; 130:3962). CD200 can identify AML cells with LSC activity in vivo (Ho et al Blood. 2016; 128:1705). Functionally, CD200 has been shown to have an immunosuppressive effect on macrophages (Hoek et al Science. 2000; 290:1768) and NK cells (Coles et al Leukemia. 2012; 26:2148), and correlates with a high prevalence of FOXP3+ regulatory T cells (Coles et al Leukemia. 2012; 26:2146). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget. 2015; 6:30212). To date, we have screened 40 primary AML patient samples by flow cytometry, 95% of which are positive for CD200. Methods: To study the functional role of CD200 in AML, we generated a CD200 overexpression model in the human OCI-AML3 cell line (with no basal expression) and characterized changes in proliferation, survival, and gene expression. To examine the immune function of CD200 in AML in vitro, we performed a series of mixed lymphocyte reactions with isolated effector immune cells and target isogenic AML cell lines to assess immune cell-mediated apoptosis, proliferation, and cytokine secretion. To understand the contribution of CD200 immune protection in a physiological setting, we developed a peripheral blood mononuclear cell (PBMC)-humanized mouse in which we tracked the engraftment and overall survival of the CD200+/- OCI-AML3 cells. Lastly, the utility of CD200-blockade using a fully humanized anti-CD200 monoclonal antibody (CD200-IgG1) was evaluated both in vitro and in vivo. Results: In vitro, CD200+ AML significantly inhibited the secretion of inflammatory cytokines and cytotoxic enzymes from healthy PBMCs; a phenomenon that could be largely reversed by blocking the CD200/CD200R interaction with the CD200 antibody (Fig 1B). In vivo, OCI-AML3 CD200+/- cells showed no difference in engraftment, progression, and overall survival in immunodeficient NSG mice (Fig 1C). However, when mice were humanized using healthy PBMCs, CD200+ leukemia progressed rapidly, escaping T cell-mediated elimination, compared to CD200- control leukemic cells (Fig 1D). Cytokine production in PBMC-humanized mice was significantly compromised in those with CD200-expressing leukemia. Transcriptome analysis revealed that T cells from humanized mice exposed to CD200 expressing disease were metabolically quiescent. In humanized mice, CD200-IgG1 therapy eliminated CD200+ AML disease (Fig 1E). The novel CD200-IgG1 antibody also induced potent, specific NK cell-mediated antibody dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody dependent cellular phagocytosis (ADCP; Fig 1F). Conclusion: We have identified CD200 as a putative stem cell-specific immunomodulatory target that aids in establishing an immunosuppressive microenvironment by significantly suppressing cytokine secretion in response to AML. In a PBMC-humanized mouse model, the presence of cell-surface CD200 was sufficient to protect AML cells from immune-mediated clearance and could be reversed using a blocking anti-CD200 mAb. These findings indicate a utility of CD200 as a novel immune checkpoint target for the development of therapeutic strategies in AML. Disclosures Konopleva: Calithera: Research Funding; Kisoji: Consultancy; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Ablynx: Research Funding; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Amgen: Consultancy; Stemline Therapeutics: Consultancy, Research Funding; AstraZeneca: Research Funding; Sanofi: Research Funding; Agios: Research Funding; Forty-Seven: Consultancy, Research Funding; Rafael Pharmaceutical: Research Funding; Ascentage: Research Funding.