scholarly journals Dasatinib Inhibits FLT3/ITD and PTPN11mutated Acute Myeloid Leukemia Cells Overexpressing SRC Tyrosine Kinases

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1451-1451
Author(s):  
Sigal Tavor ◽  
Tali Shalit ◽  
Noa Chapal Ilani ◽  
Yoni Moskovitz ◽  
Nir Livnat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Advisory Committees ◽  
Acute Myeloid

Background: Recent advances in acute myeloid leukemia(AML) targeted therapy improve overall survival. While these targeted therapies can achieve prolonged remissions, most patients will eventually relapseunder therapy. Our recent studies suggest that relapse most often originates from several sub-clones of leukemic stem cells (LSCs), present before therapy initiation, and selected due to several resistance mechanisms. Eradication of these LSCs during treatment induction /remission could thus potentially prevent relapse. The overall goal of the current study was to identify drugs which can be safely administrated to patients at diagnosis and that will target LSCs. Since simultaneously testing multiple drugs in vivo is not feasible, we used an in vitrohigh throughput drug sensitivity assay to identify new targets in primary AML samples. Methods: Drug sensitivity and resistance testing (DSRT) was assessed in vitro (N=46 compounds) on primary AML samples from patients in complete remission (N=29). We performed whole exome sequencing and RNAseq on samples to identify correlations between molecular attributes and in vitro DSRT. Results:Unsupervised hierarchical clustering analysis of in vitro DSRT, measured by IC50, identified a subgroup of primary AML samples sensitive to various tyrosine kinase inhibitors (TKIs). In this subgroup, 52% (9/17) of AML samples displayed sensitivity to dasatinib (defined as a 10-fold decrease in IC50 compared to resistant samples). Dasatinib has broad TKI activity, and is safely administered in the treatment of leukemia. We therefore focused our analysis on predicting AML response to dasatinib, validating our results on the Beat AML cohort. Enrichment analysis of mutational variants in dasatinib-sensitive and resistant primary AML samples identified enrichment of FLT3/ITD (p=0.05) and PTPN11(p=0.05) mutations among dasatinib responders. Samples resistant to dasatinib were enriched with TP53 mutations (p=0.01). No global gene expression changes were observed between dasatinib-sensitive and resistant samples in our cohort, nor in the Beat AML cohort. Following this, we tested the differential expression of specific dasatinib-targeted genes between dasatinib-responding and resistant samples. No significant differences were identified. However, unsupervised hierarchical clustering of dasatinib targeted genes expression in our study and in the Beat AML cohort identified a subgroup of AML samples (enriched in dasatinib responders) that demonstrated overexpression of three SRC family tyrosine kinases:FGR, HCK and LYN as well as PTK6, CSK, GAK and EPHB2. Analysis of the PTPN11 mutant samples revealed that the IC50 for dasatinib in 23 carriers of the mutant PTPN11 was significantly lower compared to the IC50 of PTPN11 wild type samples (p=0.005). LYN was also upregulated (p<0.001) in the mutant samples. We therefore hypothesized that gene expression of dasatinib-targeted genes could be used as a predictive biomarker of dasatinib response among FLT3/ITD carriers. We found that among FLT3/ITD AML carriers in the Beat AML cohort LYN, HCK, CSK and EPHB2 were significantly over-expressed in the dasatinib responding samples (N=27) as compared to the dasatinib resistant samples (N=35). To predict response to dasatinib among FLT3/ITD carriers we used a decision tree classifier based on the expression levels of these four genes. Our prediction model yielded a sensitivity of 74% and specificity of 83% for differentiating dasatinib responders from non-responders with an AUC of 0.84. Based on our findings, we selected FLT3/ITD AML samples and injected them to NSG-SGM3 mice. We found that in a subset of these samples, dasatinib significantly inhibited LSCs engraftment. This subset of FLT3/ITD AML samples expressed higher levels of LYN, HCK,FGR and SRC as compared to the FLT3/ITD samples that were not sensitive to dasatinib therapy in vivo. In summary, we identified a subgroup of AML patients sensitive to dasatinib, based on mutational and expression profiles. Dasatinib has anti-leukemic effects on both blasts and LSCs. Further clinical studies are needed to demonstrate whether selection of tyrosine kinase inhibitors, based on specific biomarkers, could indeed prevent relapse. Disclosures Tavor: Novartis: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS companies: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Fc Receptor-Dependent Trogocytosis of CD39 Impacts Engraftment and Invasiveness of Acute Myeloid Leukemia Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3298-3298
Author(s):  
Lili Feng ◽  
Haohai Zhang ◽  
Paola de Andrade Mello ◽  
Dina Stroopinsky ◽  
Wenda Gao ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Bioluminescence Imaging ◽  
Lentiviral Vector ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract Corresponding author: Dr. Simon. C. Robson ([email protected]). Introduction: CD39/ENTPD1 (ectonucleoside triphosphate diphosphohydrolase-1) is the prototypic member of the GDA1-CD39 superfamily of ectonucleotidases and modulates purinergic signaling pathways. CD39 expression has been noted in human acute myeloid leukemia (AML) and likely contributes to chemoresistance [1]. Our study reported here elucidates the impact of Cd39 on engraftment and invasiveness of AML TIB-49 cells using an immunocompetent murine experimental model. Methods: Wild-type (WT) mice and Cd39 -/- mice on C57BL/6 background were bred at Beth Israel Deaconess Medical Center. The syngeneic murine AML cell line TIB-49 (Cd39 negative in vitro) was purchased from American Type Culture Collection. For bioluminescence imaging experiments, TIB-49 cells were transduced with luciferase/mCherry using a lentiviral vector. For AML model, mice were administered with 1×10 6 TIB-49-luciferase cells intravenously via tail vein injection. For chloroma model, mice were subcutaneously inoculated with 1×10 6 TIB-49 cells in the right flank. Bioluminescence imaging of TIB-49-luciferase bearing mice was conducted with the IVIS TM 50 Imaging System. Blood, spleen and bone marrow (BM) were also collected from TIB-49 bearing AML mice for FACS (fluorescence activated cell sorting) analysis. To explore Cd39 in TIB engraftment and invasiveness, TIB-49 cells were further transduced with a lentiviral vector overexpressing mCd39 with TdTomato. WT mice were intravenously inoculated with 1×10 6 of either TIB-49-TdTomato cells or TIB-49-mCd39-TdTomato cells, and the above read-outs were determined. To investigate the potential of CD39 as a therapeutic target, we engineered anti-mouse Cd39 antibodies (αCd39 mAb) with isotype selection and removal of fucose to further promote Fc receptor (FcR) interactions. Results: Bioluminescence imaging results indicated that TIB-49 engraftment was decreased in global Cd39 -/- mice with decreased disease burdens noted relative to WT (Figure 1A). FACS analysis of blood, spleen and BM-derived cells from TIB-49 bearing AML-model mice (day 31) confirmed higher engraftment of TIB-49 cells (TdTomato+) at all sites in WT compared to Cd39 -/- mice (Figure 1B). TIB-49 cells did not express Cd39 in vitro, but TIB-49 cells harvested from spleen and BM of WT but not Cd39 -/- mice displayed high levels of Cd39. This indicated TIB-49 cells acquired Cd39 from host cells, in a process of antibody-independent trogocytosis (Figure 1C), as RT-PCR did not detect Cd39 mRNA expression in TIB-49 cells in vivo. Additionally, circulating TIB-49 cells from the blood of WT mice were Cd39 negative (Figure 1C), suggesting a role for the tumor microenvironment in mediating trogocytosis. TIB-49 cells expressing host Cd39 in WT mice spleen and BM lost Cd39 after being exposed to αCd39 mAb treatment. Cd39 translocated from TIB-49 cells to effector cells, at least in part, dependent on FcR mediated trogocytosis (Figure 1D). When Cd39 was overexpressed on TIB-49 cells (TIB-49-mCd39-TdTomato), the engraftment was boosted in WT mice in vivo when compared to TIB-49-TdTomato cells (day 19, Figure 1E) with higher levels of Cd39 expression than that observed on TIB-49-TdTomato cells in spleen and BM (day 26) (Figure 1F). Moreover, TIB-49-mCd39-TdTomato bearing mice displayed shorter survival times, when compared with TIB-49-TdTomato bearing AML mice (Figure 1G). The αCd39 mAb monotherapy had no effect on TIB-49 chloroma model growth. However, pretreatment with αCd39 mAb effectively boosted daunorubicin chemotherapeutic effects in vivo (Figure 1H and 1I). Conclusions: Our study suggests bidirectional trogocytosis between TIB-49 AML and host immune cells, which is further modulated by FcR interaction. Re-distribution of Cd39 from host to TIB-49 cells or induced high level expression contributes to engraftment and invasiveness, resulting in decreased survival. Targeting CD39 is a potential therapeutic approach, operational not only by boosting chemosensitivity but furthering anti-leukemic effects in experimental models. Disclosures: No relevant conflicts of interest to declare. References: [1] Nesrine Aroua, Emeline Boet, Margherita Ghisi, et al. Extracellular ATP and CD39 Activate cAMP-Mediated Mitochondrial Stress Response to Promote Cytarabine Resistance in Acute Myeloid Leukemia. Cancer Discov. 2020. Figure 1 Figure 1. Disclosures Stroopinsky: The Blackstone Group: Consultancy. Avigan: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Kite Pharma: Consultancy, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Aviv MedTech Ltd: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Legend Biotech: Membership on an entity's Board of Directors or advisory committees; Chugai: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy; Parexcel: Consultancy; Takeda: Consultancy; Sanofi: Consultancy.

scholarly journals Epigenetic Activation of the pH Regulator MCT4 in Acute Myeloid Leukemia Exploits a Fundamental Metabolic Process of Enhancing Cell Growth through Proton Shifting

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3765-3765
Author(s):  
Cheuk-Him Man ◽  
David T. Scadden ◽  
Francois Mercier ◽  
Nian Liu ◽  
Wentao Dong ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Growth ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Monocarboxylate Transporter ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership ◽  
Acute Myeloid

Acute myeloid leukemia (AML) cells exhibit metabolic alterations that may provide therapeutic targets not necessarily evident in the cancer cell genome. Among the metabolic features we noted in AML compared with normal hematopoietic stem and progenitors (HSPC) was a strikingly consistent alkaline intracellular pH (pHi). Among candidate proton regulators, monocarboxylate transporter 4 (MCT4) mRNA and protein were differentially increased in multiple human and mouse AML cell lines and primary AML cells. MCT4 is a plasma membrane H+and lactate co-transporter whose activity necessarily shifts protons extracellularly as intracellular lactate is extruded. MCT4 activity is increased when overexpressed or with increased intracellular lactate generated by glycolysis in the setting of nutrient abundance. With increased MCT4 activity, extracellular lactate and protons will increase causing extracellular acidification while alkalinizing the intracellular compartment. MCT4-knockout (MCT4-KO) of mouse and human AMLdid not induce compensatory MCT1 expression, reduced pHi, suppressed proliferation and improved animal survival. Growth reduction was experimentally defined to be due to intracellular acidification rather than lactate accumulation by independent modulation of those parameters. MCT4-KOmetabolic profiling demonstrated decreased ATP/ADP and increased NADP+/NADPH suggesting suppression of glycolysis and the pentose phosphate pathway (PPP) that was confirmed by stable isotopic carbon flux analyses. Notably,the enzymatic activity of purified gatekeeper enzymes, hexokinase 1 (HK1), pyruvate kinase M2 isoform (PKM2) and glucose-6-phosphate dehydrogenase (G6PDH) was sensitive to pH with increased activity at the leukemic pHi (pH 7.6) compared to normal pHi (pH 7.3). Evaluating MCT4 transcriptional regulation, we defined that activating histonemarks, H3K27ac and H3K4me3, were enriched at the MCT4 promoter region as were transcriptional regulators MLL1 and Brd4 by ChIP in AML compared with normal cells. Pharmacologic inhibition of Brd4 suppressed Brd4 and H3K27ac enrichment and MCT4 expression in AML and reduced leukemic cell growth. To determine whether MCT4 based pHi changes were sufficient to increase cell proliferation, we overexpressed MCT4 in normal HSPC and demonstrated in vivo increases in growth in conjunction with pHi alkalization. Some other cell types also were increased in their growth kinetics by MCT4 overexpression and pHi increase. Therefore, proton shifting may be a means by which cells respond to nutrient abundance, co-transporting lactate and protons out of the cell, increasing the activity of enzymes that enhance PPP and glycolysis for biomass generation. Epigenetic changes in AML appear to exploit that process by increasing MCT4 expression to enforce proton exclusion thereby gaining a growth advantage without dependence on signaling pathways. Inhibiting MCT4 and intracellular alkalization may diminish the ability of AML to outcompete normal hematopoiesis. Figure Disclosures Scadden: Clear Creek Bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Sponsored research; Editas Medicine: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bone Therapeutics: Consultancy; Fog Pharma: Consultancy; Red Oak Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; LifeVaultBio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics: Consultancy, Equity Ownership.

scholarly journals MEK inhibition enhances the response to tyrosine kinase inhibitors in acute myeloid leukemia

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
María Luz Morales ◽  
Alicia Arenas ◽  
Alejandra Ortiz-Ruiz ◽  
Alejandra Leivas ◽  
Inmaculada Rapado ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Resistance Pathway ◽  
Acute Myeloid

AbstractFMS-like tyrosine kinase 3 (FLT3) is a key driver of acute myeloid leukemia (AML). Several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been evaluated clinically, but their effects are limited when used in monotherapy due to the emergence of drug-resistance. Thus, a better understanding of drug-resistance pathways could be a good strategy to explore and evaluate new combinational therapies for AML. Here, we used phosphoproteomics to identify differentially-phosphorylated proteins in patients with AML and TKI resistance. We then studied resistance mechanisms in vitro and evaluated the efficacy and safety of rational combinational therapy in vitro, ex vivo and in vivo in mice. Proteomic and immunohistochemical studies showed the sustained activation of ERK1/2 in bone marrow samples of patients with AML after developing resistance to FLT3 inhibitors, which was identified as a common resistance pathway. We examined the concomitant inhibition of MEK-ERK1/2 and FLT3 as a strategy to overcome drug-resistance, finding that the MEK inhibitor trametinib remained potent in TKI-resistant cells and exerted strong synergy when combined with the TKI midostaurin in cells with mutated and wild-type FLT3. Importantly, this combination was not toxic to CD34+ cells from healthy donors, but produced survival improvements in vivo when compared with single therapy groups. Thus, our data point to trametinib plus midostaurin as a potentially beneficial therapy in patients with AML.

Expression Of Aldehyde Dehydrogenase Reflected Diverse Stem Cell Properties In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1344-1344
Author(s):  
Van T. Hoang ◽  
Eike C. Buss ◽  
Isabel Hoffmann ◽  
Abraham Zepeda-Moreno ◽  
Natalia Baran ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Clinical Study ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Aldh Activity ◽  
Advisory Committees ◽  
Low Frequencies ◽  
Acute Myeloid

Abstract Separation of leukemic stem cells (LSC) and residual hematopoietic stem cells (HSC) from the same individual patient with acute myeloid leukemia (AML) is essential for a proper understanding of the leukemic driving mechanisms. We have studied the role of aldehyde dehydrogenase (ALDH) for this purpose and have defined the functional properties of ALDHbright cells in specific subgroups of AML. We have examined the ALDH activity by flow cytometry in bone marrow samples (BM) from 14 healthy donors and 73 patients with de novo AML. The median frequency of cells with high ALDH activity (ALDHbright cells) in the healthy subjects was 1.92% with a range from 0.58 to 3.16%. For patients with AML, the median number of ALDHbright cells was 0.25% with a broad range from 0.004 to 33.57%. Whereas the majority of patients with AML (n = 56) had low frequencies of ALDHbright cells (median 0.11%; range 0.004 – 1.77%; defined as ALDH-low AML), 17 patients had relatively numerous ALDHbright cells (median 9.01; range 3.54 – 33.57%; defined as ALDH-numerous AML). In both groups, ALDHbright cell populations were highly enriched for CD34+CD38- cells. The ALDHbright cells derived from ALDH-low AML did not contain chromosomal and molecular aberrations characteristic of the original leukemia, and were able to induce multi-lineage hematopoiesis in NSG mouse models. Thus, genetically and functionally normal HSC could be successfully isolated in the ALDHbright subset, whereas LSC were enriched in ALDHdimCD34+CD38- subset for patients with ALDH-low AML. For 17 patients with ALDH-numerous AML, the ALDHbright subset was consistently contaminated with LSC. In clinical follow-ups, patients with ALDH-numerous AML showed resistance to induction chemotherapy and were characterized by a very poor long-term outcome that was comparable to patients with high-risk cytogenetic or molecular genetic markers. In four patients with ALDH-numerous AML we demonstrated that the ALDHbrightCD34+CD38- subset contained chemotherapy-resistant clones with repopulating ability. Furthermore, such ALDHbright cells were characterized by a lower cell-cycle activity and an increased resistance to cytarabine in comparison with ALDHdim blasts in in vitro assays. Our data have provided evidence that LSC and residual HSC can be separated using ALDH in patients with low frequencies of ALDHbright cells. In patients with ALDH-numerous AML, the ALDHbright subset is associated with leukemic features both in vitro and in animal models. Thus our data demonstrated the feasibility of appropriate comparisons of LSC versus HSC from the same patient with specific subtypes of AML and the impact of LSC properties on clinical outcome. Disclosures: Buss: Novartis: Travel support Other; Micromet/Amgen: Reimbursements for participation in a clinical study , Reimbursements for participation in a clinical study Other. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

scholarly journals A Novel Proteomic Profiling of the Bone Marrow Microenvironment Reveals Elevated Levels of the Chemokine CCL23 Isoforms in Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2709-2709
Author(s):  
Haydar Celik ◽  
Katherine E. Lindblad ◽  
Bogdan Popescu ◽  
Giovanna Fantoni ◽  
Gege Gui ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Serum Samples ◽  
Advisory Committees ◽  
Extracellular Compartment ◽  
Acute Myeloid

The bone marrow (BM) microenvironment is increasingly recognized as an important contributor to acute myeloid leukemia (AML) pathogenesis. However, despite growing interest in characterizing different components and cellular architecture of the BM niche and their biological significance in leukemogenesis, the proteomic constitution of the BM extracellular compartment that distinguishes a leukemic niche from its normal counterpart has not yet been fully described. We therefore performed a quantitative, large-scale proteomic analysis of 1,305 human proteins of the non-cellular compartment of BM (plasma) samples from ten relapsed or refractory AML patients and from ten age- and sex-matched healthy donors (HDs) using an aptamer-based, highly multiplexed, affinity proteomics platform (SOMAscan). This screen identified a total of 168 differentially abundant proteins, of which 91 were significantly more and 77 proteins significantly less abundant in leukemic BM compared with healthy marrow (FC ≥ 1.5, FDR ≤ 0.05). Comparative analysis of BM plasma and peripheral blood (PB) serum samples from the same AML patients and HDs revealed 65 similarly regulated proteins (37 up-regulated vs. 28 down-regulated) and 1 differently regulated protein between the two compartments. Out of the total 168 proteins, 102 proteins were specifically dysregulated only in the BM compartment. TruSeq Stranded Total RNA-sequencing (Illumina) was also performed using paired-end 75bp sequencing on a HiSeq 3000. RNA was isolated from PAXgene BM RNA tubes (Qiagen) collected in parallel with samples for proteomic analysis. Results of analysis of differentially expressed transcripts only partially overlapped with those candidates identified from our validated proteomic approach, indicating that sequencing of RNA derived from cellular sources of BM may be a suboptimal screening strategy to determine the true proteomic composition of the extracellular compartment of the AML marrow microenvironment. In addition to several previously reported proteins, our proteomics screen discovered numerous aberrantly expressed proteins in leukemic marrow whose role in AML pathogenesis is currently unknown. Using pathway analysis, we identified sets of proteins enriched for specific biological pathways including RAS, ephrin, PDGF, PI3K/AKT, MAPK, Notch, TLR, JAK-STAT, NFκB, Rap1, and Tie2 signaling pathways. A systems biology analysis approach revealed the highly connected network of cytokines and chemokines as the most striking AML-associated proteomic alteration in the BM. We identified IL-8 as a differentially expressed and key central molecule of this network in AML, consistent with recent reports. Importantly, we also identified significantly elevated levels of CKβ8 and CKβ8-1, alternatively spliced isoforms of the myelosuppressive chemokine CCL23 also known as myeloid progenitor inhibitory factor 1 (MPIF-1) or CKβ8, in both leukemic marrow and PB serum samples (Figure 1). Given the critical importance of cytopenias, often disproportional to the degree of leukemic marrow involvement, in the morbidity and mortality of patients with myelodysplastic syndrome (MDS) and AML, we subsequently confirmed this striking finding by performing orthogonal validation in a larger cohort of MDS and AML patients using an ELISA-based immunoassay. This novel finding suggests the possibility that CCL23 may play a role in suppression of normal hematopoiesis in MDS and AML. In support of this hypothesis, we demonstrated in vitro myelosuppressive effects of CCL23 isoforms on colony formation by human CD34+ hematopoietic stem and progenitor cells (HSPCs) in an in vitro colony forming unit assay, resulting in an approximately 2.5-fold decrease in CFU-GM and an evident decrease in CFU-GEMM counts. In summary, our broad and quantitative proteomic dataset of extracellular factors present in leukemic and normal aging bone marrow has already provided novel mechanistic insights into AML pathogenesis and should serve, together with paired RNA-sequencing information, as a useful public resource for the research community. Disclosures Lai: Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Speakers Bureau; Astellas: Speakers Bureau; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees. Hourigan:SELLAS Life Sciences Group AG: Research Funding; Merck, Sharpe & Dohme: Research Funding.

scholarly journals The Long Noncoding RNA BALR2 Controls Novel Transcriptional Circuits Involved in Chemotherapy Sensitivity of Pediatric Acute Myeloid Leukemia (AML) Blasts

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2734-2734
Author(s):  
Valeria Bisio ◽  
Maddalena Benetton ◽  
Elena Porcù ◽  
Matteo Bordi ◽  
Carlo Zanon ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Board Of Directors ◽  
Expression Profile ◽  
Myeloid Leukemia ◽  
Stem Cell Differentiation ◽  
Myeloid Differentiation ◽  
Advisory Committees ◽  
Acute Myeloid

In acute myeloid leukemia (AML), the assessment of post-induction minimal residual disease (MRD) is largely utilized for choosing post-remission therapies aimed at maintaining complete remission (CR) and preventing relapse. This latter is still the major cause of treatment failure in pediatric AML, and even if several efforts have been spent to validate MRD as a prognostic marker, numerous studies demonstrated that MRD negativity cannot be considered a completely reliable surrogate biomarker predicting outcome, since it does not exclude a relapse. The current interpretation is that disease relapse is due to mechanisms leading to therapy resistance mainly depending on driver chimeric or oncogenic protein-coding genes, which are monitored during treatment, and does not consider that chemotherapy resistance may arise from other genetic markers, phenomenon linked to methylation and non-coding RNAs genomic pressure. We, thus, hypothesized that other markers need to be explored to re-interpret leukemia progression. We showed an overall hyper-expression of the lncRNA BALR2 in 132 de novo AML bone marrow samples collected at diagnosis and analyzed the gene expression profile (GEP) of 58 cases. By unsupervised clustering analysis, we produced important advances in identifying BALR2 as a robust novel molecular marker of a new subgroup of AML characterized by a high rate of resistance to induction therapy, independently from the genetic lesions detected at diagnosis and any other prognostic clinical and genetic features. We demonstrated in vitro that BALR2 has a direct role in controlling bi-directionally its own and of its neighbor gene CDK6 promoter activity. This latter finding of high CDK6 expression was shown to sustain its complex with RUNX1 in order to inhibit RUNX1 binding to its target promoters, thus preventing the process of hematopoietic differentiation progression. To support BALR2 as a new proto-oncogene involved in the control of the myeloid differentiation program, we ranked the genes across the expression profile obtaining a signature of 337 transcripts able to cluster CD34+ human stem cell precursors (HSCPs) separately from more mature CD14+ cells. These in silico findings were validated in vitro by showing that, after BALR2 depletion, CD34+ cells had a skewed myeloid differentiation. Furthermore, we found that AML differentiation toward mature myeloid cells with increased phagocytic capacity was obtained through BALR2 level reduction, and enhanced by combinatorial differentiation stimuli. Our findings attribute a distinct role to BALR2 in the block of myeloid stem cell differentiation occurring during leukemogenesis. At the same time, we interrogated GEP ontology, finding that enrichments of genes involved in mitochondrial synthesis pathways were significantly correlated to patients with highest BALR2 levels, and confirmed the same mitochondriogenesis profile in the immature CD34+ HSCPs. We moved to deconvolute this feature and demonstrated that BALR2, by controlling mitochondria gene balance, was directly controlling the mitochondrial mass, which dramatically decreased after BALR2 silencing, this supporting the hypothesis that BALR2 would maintain mitochondrial functions to confer AML resistance to cytotoxicity. Consistently with this line of reasoning, we inhibited mitochondria by tigecycline, demonstrating that its activity was dramatically strengthened in BALR2 depleted cells, when used either alone or in combination with cytosine-arabinoside (Ara-C). Concomitantly, tigecycline treatment in BALR2 silenced AML cells reduced mitochondria depolarization, and increased the number of differentiated M-CFU colonies formation, confirming that BALR2, together with CDK6, forms novel transcriptional networks to create a circuit able to impair myeloid differentiation and to lower chemo-sensitivity in AML. We speculate that a novel therapeutic window of mitochondrial targeting in defined AML subgroups, identified through assessment of BALR2 levels at diagnosis or persistent MRD levels, could be envisaged to optimize the outcome of childhood AML. Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.

scholarly journals RORA Is a Potential Prognostic Biomarker and Therapeutic Target for Patients with Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2696-2696
Author(s):  
Christina A. Snider ◽  
Kevin Fung ◽  
Francesca Gould ◽  
Vera Adema ◽  
Cassandra M Kerr ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Growth Inhibition ◽  
Mrna Expression ◽  
Board Of Directors ◽  
Therapeutic Target ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Advisory Committees ◽  
Acute Myeloid

One strategy to improve precision medicine in acute myeloid leukemia (AML) is to further our understanding of the biological factors that influence pharmacologic efficacy. In a recent study, whole transcriptome analysis was conducted using pre- and post-treatment samples from a cohort of relapsed or refractory (R/R) AML patients treated with mitoxantrone, etoposide, cytarabine (MEC), and ixazomib. Logistic regression and linear discriminant analysis identified RORa as a predictor of response to treatment (Advani, et al., Clin Cancer Res, 2019, 4231-4237). RORa is not frequently mutated in AML but is described to be a tumor suppressor in patients with solid tumors. In the latter patient population, increased RORa expression is also associated with improved survival. We first retrospectively evaluated the prognostic significance of RORa in a larger cohort of R/R patients with AML (BeatAML). Second, we characterized RORa expression in cellular models of AML and sought to determine whether a commercially available RORa/RORγ agonist, SR1078, has anti-proliferative capacity in leukemia cell lines. For the correlative survival analyses, RORa mRNA expression and clinical information was downloaded from the BeatAML cohort (Tyner, et al., Nature, 2018, 526-531). Patients were categorized into high expressers (≥ median) and low expressers (<median). In total, 121 R/R patients were investigated. The mean age of diagnosis was 62 ± 12 years, and 58% of patients were male. The most common specific diagnosis at inclusion was AML with myelodysplasia related changes (28%), followed by AML with mutated NPM1 (18%) and therapy-related myeloid dysplasia (13%). Over half of the analyzed samples were peripheral blood (58%), and the remaining samples were either bone marrow aspirate (58%) or from leukapharesis (2%). As a whole, the median mRNA expression levels of RORa in patients with R/R AML (n=121) compared to healthy subjects (n=21) were 6.6 log2 CPM vs. 2.5 log2 CPM (P<.0001). After grouping patients into low RORa expresser and high RORa expresser groups, patients with above median expression of RORa were found to have significantly longer overall survival (19 mo. vs. 13 mo.; P=.0052; Figure 1). During normal hematopoiesis, we observed that RORa expression decreased with the stages of cellular maturation and is highly expressed in cells of AML patients with complex karyotype (BloodSpot, 2018). We noted that RORa mRNA expression varied across patients suggesting differences between AML subtypes. Expression levels were also confirmed to be different across AML subtypes in vitro by using cell line models (K-562, KG-1, OCI-AML3, U-937, THP-1, MOLM-13). In particular, analysis of TCGA data showed higher mean RORa expression in DNMT3A mutant (MT) (626 RPKM) compared to mean RORa expression in CEBPAMT (243 RPKM; P=0.026), NPM1MT (167 RPKM; P=0.012), and FLT3MT (236 RPKM; P=.003) AML patients. We then analyzed the sensitivity of cell lines to the commercially available synthetic RORa /RORγ ligand, SR1078. Myeloid lineage cells U-937 and KG-1 were used as a cellular model. Cells in logarithmic phase were treated with increased concentrations of the RORa /RORγ agonist SR1078 (from 3 nM to 30 mM) for 24 hours. Cell viability was measured by MTT tetrazolium reduction assay. Maximal growth inhibition was reached at 30 µM for U-937 (80%) and KG-1 (75%), respectively. We then combined SR1078 with MEC to evaluate whether in vitro SR1078 increased MEC growth inhibition. The addition of SR1078 to MEC significantly decreased cell viability in KG-1 cells compared to MEC alone (82% vs. 19%, P=.029). Our study suggests that increased RORa expression may be associated with improved survival in patients with R/R AML and that RORa may be a potential therapeutic target in AML. Figure 1 Disclosures Gerds: Celgene Corporation: Consultancy, Research Funding; Imago Biosciences: Research Funding; Pfizer: Consultancy; CTI Biopharma: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Roche: Research Funding; Sierra Oncology: Research Funding. Mukherjee:Bristol-Myers Squibb: Speakers Bureau; Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Partnership for Health Analytic Research, LLC (PHAR, LLC): Consultancy; McGraw Hill Hematology Oncology Board Review: Other: Editor; Projects in Knowledge: Honoraria. Nazha:Jazz Pharmacutical: Research Funding; Incyte: Speakers Bureau; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Tolero, Karyopharma: Honoraria; Abbvie: Consultancy; Daiichi Sankyo: Consultancy. Maciejewski:Alexion: Consultancy; Novartis: Consultancy. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees. Advani:Abbvie: Research Funding; Pfizer: Honoraria, Research Funding; Glycomimetics: Consultancy, Research Funding; Amgen: Research Funding; Macrogenics: Research Funding; Kite Pharmaceuticals: Consultancy.

scholarly journals Chloroquine Derivative Lys05 Overcomes Hypoxia-Induced Chemoresistance in Acute Myeloid Leukemia through Metabolic Disruption

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3948-3948
Author(s):  
Matthew Johnson ◽  
Kaitlyn Dykstra ◽  
Tara Cronin ◽  
Linda Lutgen-Dunckley ◽  
Brandon L Martens ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Late Stage ◽  
Myeloid Leukemia ◽  
Tumor Burden ◽  
Advisory Committees ◽  
Mitochondrial Mass ◽  
Metabolic Disruption ◽  
Acute Myeloid

Abstract Background: Despite initial responses to standard chemotherapy, most patients with acute myeloid leukemia (AML) relapse and have poor prognoses after subsequent therapy. The hypoxic bone marrow (BM) microenvironment is hypothesized to contribute to clinical resistance through the sheltering of AML blasts and leukemia stem cells (LSCs), allowing them to evade chemotherapy and reinitiate the disease. We have previously shown that AML cell lines cultured under prolonged hypoxia (1% O2) can upregulate autophagy and become resistant to cytarabine (AraC). Furthermore, the inhibition of autophagosome turnover with either Bafilomycin A1 (Baf A1) or chloroquine (CQ) can re-sensitize AML cells to AraC. Additionally, Baf A1 therapy effectively eradicated in vivo functional LSCs in a primary human AML xenotransplantation mouse model. However, clinical development of BafA1 and CQ has been limited by poor drug pharmokinetics. Lys05 is a novel water-soluble CQ derivative which exhibits 10 fold more potent autophagy inhibitory properties than CQ in vitro and is well tolerated in mouse models. Here, we tested the effects of Lys05 on human AML growth, chemoresistance under hypoxia, and in vivo leukemia burden in a human AML xenograft model. We also explored the effects of autophagosome accumulation on cellular metabolism as a new mechanism for targeting chemoresistant AML blasts and LSCs. Methods: Human AML cell line MOLM13 and CD34 positive AML patient samples were cultured under hypoxia (1% O2) or normoxia (21% O2) with cytarabine (AraC) and late-stage autophagy inhibitor Lys05 alone and in combination. After 72 hours of drug exposure, apoptosis was measured using Annexin V/propidium iodide flow cytometry. Metabolic assays were performed using a Seahorse XFe96 analyzer to measure oxygen consumption and extracellular acidification rates using the Mitochondrial or Glycolytic Stress Tests on AML cells treated with late-stage autophagy inhibitors for 48 hours. Mitochondrial mass was measured following staining with MITO-ID Green Detection Kit using flow cytometry. Human xenograft models were performed using NSG mice inoculated with MOLM13 BLIV, a luciferase-expressing human AML cell line, and treated with either vehicle or Lys05 (20 mg/kg 4 days on/3 off for 4 weeks). Tumor burden was assessed using bioluminescence and analyzed with IVIS Living Image Software. Results: Treatment of AML MOLM13 cells with Lys05 in vitro resulted in a dose-dependent decrease in cell proliferation and increased apoptosis. Lys05 treatment further displayed similar results to BafA1 and CQ, enhancing the anti-leukemic effects of AraC and overcoming hypoxia-induced chemoresistance. Seahorse XFe analysis revealed that BafA1 inhibited both basal and maximal mitochondrial respiration under both normoxic and hypoxic conditions at 24 hours. CQ and Lys05, however, had no effect on oxidative phosphorylation under normoxia, yet significantly decreased mitochondrial function under hypoxia. Interestingly, reduced function did not coincide with a decrease in mitochondrial mass, but rather increased mitochondrial mass. We also observed decreases in glycolytic function after treatment with late-stage autophagy inhibitors under hypoxia suggesting these cells may be undergoing an energy crisis. Treatment of leukemia-bearing mice with Lys05 trended toward decreased tumor burden compared to vehicle. Studies optimizing Lys05 monotherapy and testing combination treatment with AraC are underway. Conclusions: Our research shows that CQ derivative Lys05 exhibits significant anti-leukemic activity against human AML cells and restores chemosensitivity to cytarabine under hypoxia, further supporting our hypothesis that autophagosome accumulation can overcome hypoxia-induced chemoresistance. We also demonstrate that Lys05 may effectively inhibit leukemia progression in vivo. Our studies further reveal metabolic disruption through the accumulation of autophagosomes as a mechanism for restoring chemosensitivity of AML cells under hypoxia and targeting LSCs. These results establish late-stage autophagy inhibitors, specifically Lys05, as a promising new treatment approach for resistant AML and supports further study of these drugs for treatment of minimal residual disease. Disclosures Guzman: Cellectis: Research Funding. Wang:Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Amgen: Consultancy; Jazz: Speakers Bureau.

scholarly journals Associating Ex Vivo Drug Sensitivity with Differentiation Status Identifies Effective Drug Combinations for Acute Myeloid Leukemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1301-1301
Author(s):  
Christopher A. Eide ◽  
Stephen E. Kurtz ◽  
Andy Kaempf ◽  
Nicola Long ◽  
Daniel Bottomly ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Board Of Directors ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Drug Sensitivity ◽  
Ex Vivo ◽  
Single Agent ◽  
Publicly Traded ◽  
Advisory Committees ◽  
Acute Myeloid

Abstract The development of molecularly-targeted therapies to improve outcomes relative to chemotherapy for acute myeloid leukemia (AML) is impeded by the heterogeneity of genetic aberrations that contribute to disease. Among the multitude of biological mechanisms that lead to AML disease initiation and progression is dysregulation of cytokine signaling pathways, a hallmark of chronic inflammation, which contribute to the growth, survival, and differentiation state of AML cells. We have previously shown that IL-1β, a pro-inflammatory cytokine expressed by many cell types including macrophages and monocytes, stimulates proliferation of leukemic blasts independent of mutational status in primary AML samples via enhanced phosphorylation of p38α MAPK, an effect that can be blocked by IL-1 receptor knockdown or by pharmacologic inhibition (Carey 2017). Additionally, recent studies have shown sensitivity to the approved BCL2 inhibitor venetoclax in AML associates with undifferentiated leukemic cells (Pei 2020; Zhang 2018; Majumder 2020). Based on these associations, we evaluated the combination of doramapimod (DORA), a p38 MAPK inhibitor, with venetoclax (VEN) for potential enhanced sensitivity on primary AML cells. Ex vivo drug screening of primary AML patient samples (n=335) revealed significantly enhanced efficacy of VEN+DORA compared to either single agent (Nemenyi test; p&lt;0.0001). This broad sensitivity of the VEN+DORA combination was not significantly associated with an array of clinical, genetic, and mutational features in the patient samples tested, in contrast to single agents, particularly VEN. Analysis of blood cell differential counts of patient samples tested identified increased monocyte levels were significantly correlated with sensitivity to DORA and resistance to VEN as single agents (Spearman r = -0.3 and 0.6; p&lt;0.0001), associations that were not apparent with the combination. For patient samples with accompanying FAB differentiation state-based designations (n=108), sensitivities of the combination were similar across classifications of undifferentiated (M0/M1) through monocytic (M4/M5) acute leukemia. In contrast, single-agent VEN was significantly more sensitive in undifferentiated compared to monocytic specimens, whereas DORA sensitivity showed the reverse trend (though to a lesser degree). These differences in sensitivity were further validated by immunophenotyping data where available (n=105), which showed surface markers associated with resistance to VEN (CD11b, CD14, CD16, CD56, CD64, HLADR; Wilcoxon Rank Sum, p&lt;0.001 to p=0.007) or sensitivity to VEN (CD117; p=0.001) or DORA (CD14; HLADR; p=0.004). By contrast, none of these associations significantly distinguished sensitivity for the VEN+DORA combination. Expression levels of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were concordant with their respective drug sensitivities associated with FAB classification; that is, significantly higher levels of BCL2 in M0/M1 leukemias and MAPK14 in M4/M5 cases (Mann-Whitney test; p&lt;0.0001; n=145). Further dissection of transcriptomic and drug sensitivity data revealed strong correlation and gene set enrichment for DORA and VEN sensitivities with monocyte-like and progenitor-like signatures, respectively (n=225), for cell differentiation states previously described for AML (van Galen 2019), and these associations diminished for the combination treatment. Lastly, the VEN+DORA combination enhanced efficacy and synergistic inhibition was confirmed using human AML cell line models tested with a matrix of potential dose concentrations. Taken together, these findings suggest that exploiting distinct, complementary sensitivity profiles of targeted therapies with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers an opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML. Disclosures Druker: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Recludix Pharma, Inc.: Consultancy; EnLiven: Consultancy, Research Funding; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Aileron: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Genentech: Research Funding; Takeda: Research Funding; Astrazeneca: Research Funding; Constellation: Research Funding; Agios: Research Funding; Petra: Research Funding; Incyte: Research Funding; Array: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding.

scholarly journals Potentially Curative Therapeutic Activity of NEO212, a Perillyl Alcohol-Temozolomide Conjugate, in Preclinical Cytarabine-Resistant Models of Acute Myeloid Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3385
Author(s):  
Axel H. Schönthal ◽  
Steve Swenson ◽  
Radu O. Minea ◽  
Hye Na Kim ◽  
Heeyeon Cho ◽  
...  
Keyword(s):  
Cell Death ◽  
Acute Myeloid Leukemia ◽  
Side Effects ◽  
Anticancer Activity ◽  
Myeloid Leukemia ◽  
Perillyl Alcohol ◽  
Therapeutic Activity ◽  
Acute Myeloid

Despite progress in the treatment of acute myeloid leukemia (AML), the clinical outcome remains suboptimal and many patients are still dying from this disease. First-line treatment consists of chemotherapy, which typically includes cytarabine (AraC), either alone or in combination with anthracyclines, but drug resistance can develop and significantly worsen prognosis. Better treatments are needed. We are developing a novel anticancer compound, NEO212, that was created by covalent conjugation of two different molecules with already established anticancer activity, the alkylating agent temozolomide (TMZ) and the natural monoterpene perillyl alcohol (POH). We investigated the anticancer activity of NEO212 in several in vitro and in vivo models of AML. Human HL60 and U937 AML cell lines, as well as different AraC-resistant AML cell lines, were treated with NEO212 and effects on cell proliferation, cell cycle, and cell death were investigated. Mice with implanted AraC-sensitive or AraC-resistant AML cells were dosed with oral NEO212, and animal survival was monitored. Our in vitro experiments show that treatment of cells with NEO212 results in growth inhibition via potent G2 arrest, which is followed by apoptotic cell death. Intriguingly, NEO212 was equally potent in highly AraC-resistant cells. In vivo, NEO212 treatment strikingly extended survival of AML mice and the majority of treated mice continued to thrive and survive without any signs of illness. At the same time, we were unable to detect toxic side effects of NEO212 treatment. All in all, the absence of side effects, combined with striking therapeutic activity even in an AraC-resistant context, suggests that NEO212 should be developed further toward clinical testing.

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