scholarly journals SYK inhibition targets acute myeloid leukemia stem cells by blocking their oxidative metabolism

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Anna Polak ◽  
Emilia Bialopiotrowicz ◽  
Beata Krzymieniewska ◽  
Jolanta Wozniak ◽  
Marta Stojak ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Mitochondrial Biogenesis ◽  
Oxidative Metabolism ◽  
Cell Biology ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Metabolic Reprogramming ◽  
Therapeutic Modality ◽  
Acute Myeloid

Abstract Spleen tyrosine kinase (SYK) is an important oncogene and signaling mediator activated by cell surface receptors crucial for acute myeloid leukemia (AML) maintenance and progression. Genetic or pharmacologic inhibition of SYK in AML cells leads to increased differentiation, reduced proliferation, and cellular apoptosis. Herein, we addressed the consequences of SYK inhibition to leukemia stem-cell (LSC) function and assessed SYK-associated pathways in AML cell biology. Using gain-of-function MEK kinase mutant and constitutively active STAT5A, we demonstrate that R406, the active metabolite of a small-molecule SYK inhibitor fostamatinib, induces differentiation and blocks clonogenic potential of AML cells through the MEK/ERK1/2 pathway and STAT5A transcription factor, respectively. Pharmacological inhibition of SYK with R406 reduced LSC compartment defined as CD34+CD38−CD123+ and CD34+CD38−CD25+ in vitro, and decreased viability of LSCs identified by a low abundance of reactive oxygen species. Primary leukemic blasts treated ex vivo with R406 exhibited lower engraftment potential when xenotransplanted to immunodeficient NSG/J mice. Mechanistically, these effects are mediated by disturbed mitochondrial biogenesis and suppression of oxidative metabolism (OXPHOS) in LSCs. These mechanisms appear to be partially dependent on inhibition of STAT5 and its target gene MYC, a well-defined inducer of mitochondrial biogenesis. In addition, inhibition of SYK increases the sensitivity of LSCs to cytarabine (AraC), a standard of AML induction therapy. Taken together, our findings indicate that SYK fosters OXPHOS and participates in metabolic reprogramming of AML LSCs in a mechanism that at least partially involves STAT5, and that SYK inhibition targets LSCs in AML. Since active SYK is expressed in a majority of AML patients and confers inferior prognosis, the combination of SYK inhibitors with standard chemotherapeutics such as AraC constitutes a new therapeutic modality that should be evaluated in future clinical trials.

scholarly journals 852 Trifunctional NKp46/CD16a-NK cell engager targeting CD123 overcomes acute myeloid leukemia resistance to ADCC

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A893-A893
Author(s):  
Laurent Gauthier ◽  
Angela Virone-Oddos ◽  
Angela Virone-Oddos ◽  
Jochen Beninga ◽  
Benjamin Rossi ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Antitumor Activity ◽  
Nk Cells ◽  
Myeloid Leukemia ◽  
Nk Cell ◽  
Ex Vivo ◽  
Activating Receptors ◽  
Acute Myeloid

BackgroundThere is a clear need for targeted therapies to treat acute myeloid leukemia (AML), the most common acute leukemia in adults. CD123 (IL-3 receptor alpha chain) is an attractive target for AML treatment.1 However, cytotoxic antibody targeting CD123 proved insufficiently effective in a combination setting in phase II/III clinical trials.2 T-cell engagers targeting CD123 displayed some clinical efficacy but were often associated with cytokine release syndrome and neurotoxicity.3 Interest in the use of NK cells for therapeutic interventions has increased in recent years, as a potential safer alternative to T cells. Several NK-cell activating receptors, such as CD16a, NKG2D, and the natural cytotoxicity receptors NKp30 and NKp46, can be targeted to induce antitumor immunity. We previously reported the development of trifunctional NK-cell engagers (NKCEs) targeting a tumor antigen on cancer cells and co-engaging NKp46 and CD16a on NK cells.4MethodsWe report here the design, characterization and preclinical development of a novel trifunctional NK cell engager (NKCE) targeting CD123 on AML cells and engaging the activating receptors NKp46 and CD16a on NK cells. The CD123 NKCE therapeutic molecule was engineered with humanized antibodies targeting NKp464 and CD123.5 We compared CD123-NKCE and a cytotoxic ADCC-enhanced antibody (Ab) targeting CD123, in terms of antitumor activity in vitro, ex vivo and in vivo. Pharmacokinetic, pharmacodynamic and safety profile of CD123-NKCE were evaluated in non-human primate (NHP) studies.ResultsThe expression of the high affinity Fc gamma receptor CD64 on patient-derived AML cells inhibited the ADCC of the Ab targeting CD123 in vitro and ex vivo, but not the antitumor activity of CD123-NKCE. CD123-NKCE had potent antitumor activity against primary AML blasts and AML cell lines, promoted strong NK-cell activation and induced cytokine secretion only in the presence of AML target cells. Its antitumor activity in mouse model was greater than that of the comparator antibody. Moreover, CD123-NKCE had strong and prolonged pharmacodynamic effects in NHP when used at very low doses, was well-tolerated up to high 3 mg/kg dose and triggered only minor cytokine release.ConclusionsThe data for activity, safety, pharmacokinetics, and pharmacodynamics provided here demonstrate the superiority of CD123-NKCE over comparator cytotoxic antibody, in terms of antitumor activity in vitro, ex vivo, in vivo, and its favorable safety profile, as compared to T-cell therapies. These results constitute proof-of-principle for the efficacy of CD123-NKCE for controlling AML tumors in vivo, and provide consistent support for their clinical development.ReferencesEhninger A, Kramer M, Rollig C, et al. Distribution and levels of cell surface expression of CD33 and CD123 in acute myeloid leukemia. Blood Cancer J 2014;4:e218.Montesinos P, Gail J Roboz GJ, et al. Safety and efficacy of talacotuzumab plus decitabine or decitabine alone in patients with acute myeloid leukemia not eligible for chemotherapy: results from a multicenter, randomized, phase 2/3 study. Leukemia 2021;35(1):62–74.Uy GL, Aldoss I, Foster MC, et al. Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia. Blood 2021;137(6):751–762.Gauthier L, Morel A, Anceriz N, et al. Multifunctional natural killer cell engagers targeting NKp46 trigger protective tumor immunity. Cell 2019;177(7):1701–13.Jin L, Lee EM, Ramshaw HS, et al. Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 2009;5:31–42.

scholarly journals Multi-Antigen Primed T Cells Promote Apoptosis of Acute Myeloid Leukemia (AML)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4794-4794
Author(s):  
Ebtesam Nafie ◽  
Mathias Oelke ◽  
Melissa Valerio ◽  
Sojung Kim ◽  
Ivan Rodriguez ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Time Dependent ◽  
Target Cells ◽  
Ifn Γ ◽  
Acute Myeloid

Abstract Introduction Acute myeloid leukemia (AML), the most common acute leukemia in adults, is characterized by uncontrolled proliferation of immuature myeloid cells. Despite newly approved drugs, AML remains largely incurable due to the persistence of the leukemia stem cell (LSC) population which lie quiescent in the bone marrow niche. Immunotherapy has potential to eradicate LSCs, however, no unique LSC immunophenotype has been identified. Moreover, it is necessary to simultaneously target multiple antigens (Ags) to prevent immune escape and to overcome refractory disease. We present in vitro studies in support of a therapeutic platform capable of targeting multiple intracellular Ags which could meet this challenge. The adoptive transfer of activated T cells primed to engage diverse AML associated epitopes by ex vivo exposure to artificial Ag presenting cells (aAPC) has the potential to eliminate both primary leukemia blasts and LSCs. Hypothesis Ex vivo enrichment and expansion (E+E) of antigen-educated CD8+ T cells recognizing 5 peptides derived from 3 proteins, Cyclin A1, PRAME and WT1, can selectively identify, engage, and kill AML cell lines or patient-derived (PD) AML blasts in a HLA A*02:01 restricted manner in vitro. Materials and Methods T cells from the peripheral blood mononuclear cell fraction of a healthy HLA A*02:01 donor were enriched for antigen-educated CD8+/CD4 -T cells. These cells were cultured with nanoparticles decorated with the 5 peptides and a costimulatory protein, resulting in the activation and expansion of those T cells expressing the cognate T cell receptors. These cells are composed of ~97% abT cells, 3% gdT cells and ~13% T scm, 41.5% T cm, 39.5%T em, 6%T emra and 1% T n. Results Ex vivo expanded educated T cells exhibit target-specific anti-AML activity. T cell mediated cell apoptosis of HLA-matched THP1 cells is dose and time-dependent. At 10:1 effector to target (E:T) ratio, ~28% apoptosis occurred at 24 hrs, while apoptosis was at basal levels when antigen non-educated T cells were used (data not shown). Studies were extended to PD AML cells (Fig. 1A: 012; Fig. 1B: 415) where antigen educated T cells elicited rapid (<16 hrs) and extensive (~90%) apoptosis of target PD AML cells at all E:T ratios examined. Time lapse photography of T cell/PD AML incubations revealed antigen-educated T cells clustered around AML cells (Fig. 2A), a fraction of the latter disappearing over the course of 12 hrs while PD AML cells incubated with non-educated T cells (Fig. 2B) remained viable over 12 hrs. Furthermore, there is little or no T cell movement or clustering in the wells with unprimed, non-active T cells. Release of IFN-γ by educated T cells. T cells (Fig.3A: antigen-educated through E+E) were incubated at E:T::5:1 for 24 to 48 hrs and IFN-γ in supernatants measured. The fold difference over non-educated T cells incubated with AML cells for the same time is shown and can reach over 5-fold. IFN-γ accumulation was time-dependent where antigen-educated T cells were combined with HLA-A2 matched THP1 or PD AML cells (012, 415, 470). Educated T cells were not active against target cells lacking HLA-A2 (K562) demonstrating HLA restricted killing (Fig. 3B). Additionally, antigen-educated T cells incubated without any target released slightly more IFN-γ than non-educated T cells under similar conditions but AML cells fail to stimulate IFN-γ release from non-educated T cells (data not shown). Conclusions We demonstrate HLA restricted cytotoxic activity of antigen-educated T cells against THP1 AML cells and PD AML blasts as shown by flow cytometry and microscopy. Consistent with target cell death, the supernatants from assays with antigen-educated T cells and HLA A*02:01 AML target cells exhibited over 5-fold more IFN-γ than media from assays of non-educated cells under identical conditions. Under these in vitro conditions, PD AML blasts were more readily killed than THP1 cells perhaps due to higher target antigen density (data not shown). These results support the use of multi-antigen-educated T cells for adoptive transfer to treat AML. To investigate the safety and establish the recommended phase II dose, a multi-center Phase I clinical study is underway in relapsed AML post-allo-HCT (NCT 04284228). Future studies will incorporate new antigens to enable broader targeting of a heterogeneous population of AML within and across patients Figure 1 Figure 1. Disclosures Oelke: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Marcucci: Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings. Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Hansa Biopharma: Consultancy; Neximmune: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy.

scholarly journals Evaluation of a Bifunctional Sirpα-CD123 Fusion Antibody for the Elimination of Acute Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2544-2544 ◽  
Author(s):  
Siret Tahk ◽  
Saskia Schmitt ◽  
Christian Peter Augsberger ◽  
Binje Vick ◽  
Laia Pascual Ponce ◽  
...  
Keyword(s):  
Stem Cells ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Regulatory Protein ◽  
Target Antigen ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Background: Despite considerable advances in the development of novel strategies for the treatment of acute myeloid leukemia (AML) the relapse rate is still high with only limited treatment options. Relapse occurs due to the persistence of chemotherapy-resistant leukemic stem cells (LSCs), which re-initiate outgrowth of the disease, highlighting the need of targeting LSCs to improve overall survival. Immunotherapies represent a promising strategy to target chemotherapy-resistant LSCs in AML. LSCs are characterized by the expression of the interleukin-3 receptor α, also known as CD123. CD123 is expressed on AML blasts and LSCs, and shows only a moderate expression on normal hematopoietic stem cells, claiming CD123 as a suitable target antigen (Haubner et al, Leukemia 2019). CD47, known as a marker of self, is also highly expressed on LSCs as immune escape mechanism. CD47 transmits a "don't eat me" signal upon its interaction with the myeloid-specific signal regulatory protein alpha (SIRPα) receptor on macrophages, thus inhibiting phagocytosis. In order to efficiently eliminate LSCs and provide AML patients a possibility for prolonged relapse-free survival, we have designed a bifunctional antibody that specifically targets CD123 and simultaneously blocks CD47. Importantly, our strategy restricts the benefits of the CD47 blockade to CD123 positive AML cells. Thus, we hypothesize a lower risk for on-target off-leukemia toxicity. Methods: The bifunctional SIRPα-CD123 antibody was generated by fusing the endogenous extracellular domain of SIRPα, which functions as the CD47 blocking domain, to an CD123 antibody CD123. We assessed the selective binding of the bifunctional antibody to CD123+CD47+ AML-derived cells and the ability to block CD47 on CD123+ cells in vitro. Furthermore, the biological activity of the SIRPα-CD123 antibody was examined using the AML-derived cell line MOLM-13, patient-derived xenografted (PDX) AML cells as well as primary cells from patients with newly diagnosed or relapsed AML. Results: We engrafted the endogenous SIRPα V-like domain to an antibody targeting CD123, which improved the binding of the bifunctional SIRPα-CD123 antibody to AML cells compared to a conventional CD123 antibody (MFI ratioCD123 = 2.46 0.25 vs MFI ratioSIRPα-CD123 = 4.44 0.60). The SIRPα-CD123 antibody enhanced the elimination of the AML-derived MOLM-13 cells by antibody-dependent cellular cytotoxicity (EC50CD123 = 38.5 pM vs EC50SIRPα-CD123 = 10.1 pM, n = 9). Additionally, the cytotoxicity was confirmed using primary patient-derived AML cells ex vivo. Further, an improved ex vivo cytotoxicity towards AML PDX cells was observed with the SIRPα-CD123 antibody (% lysis at 100 nM: 14.27 5.40 vs 42.94 10.21 for CD123 and SIRPα-CD123 antibodies respectively, n = 3). With regards to the inhibition of CD47 signaling, we were able to show a blockade of CD47 on CD123+CD47+ positive cells by the SIRPα-CD123 antibody. Correspondingly, a significant increase in phagocytosis of primary patient-derived AML cells mediated by monocyte-derived macrophages was observed in allogenic as well as autologous settings (% phagocytosis, normalized to isotype control and maximum phagocytosis in an autologous setting: 20.11 4.59 vs 90.37 6.22, n = 5 for CD123 and SIRPα-CD123 antibodies, respectively). We were further able to show a preferential binding to MOLM-13 in the presence of a 20-fold excess of red blood cells indicating a potential low on-target off-leukemia toxicity. Taken together, our in vitro data supports the elimination of the CD123+CD47+ positive AML LSC compartment by a synergistic effect of avidity-dependent binding to CD123 and CD47 and the simultaneous inhibition of the innate immune CD47-SIRPα signaling pathway. Conclusions: The SIRPα-CD123 is a bifunctional antibody with the potential to deplete CD123+CD47+ AML LSCs by a dual mode of action mechanism resulting in NK cell dependent cytotoxicity and macrophage-mediated phagocytosis. By combining a high affinity binding to CD123+ cells and a low affinity CD47 blockade that is restricted to CD123+ cancer cells we effectively minimize the risk for CD47-related on-target off-leukemia toxicity. The results of our in vitro assays using AML cell lines are consistent with the data from PDX and primary AML samples and support further preclinical testing of the SIRPα-CD123 antibody in vivo. Disclosures Subklewe: Miltenyi: Research Funding; Pfizer: Consultancy, Honoraria; Gilead: Consultancy, Honoraria, Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Oxford Biotherapeutics: Research Funding; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria; Morphosys: Research Funding; Janssen: Consultancy.

TCR and DNAM Dependent Lysis of Acute Monocytic Leukemia (AMoL) by Vg9Vd2 T Lymphocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1041-1041
Author(s):  
Julie Gertner-Dardenne ◽  
Eloise Perrot ◽  
Thomas Prebet ◽  
Aude Charbonnier ◽  
Helene Sicard ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
T Lymphocytes ◽  
Myeloid Leukemia ◽  
Ex Vivo ◽  
Acute Myeloid

Abstract Abstract 1041 Poster Board I-63 BACKGROUNd: Compelling evidences have demonstrated the role of the immune system in the control of acute myeloid leukemia (AML). So far, T cells and natural killer (NK) cells are the major immune effectors shown to be involved in AML control. The graft-versus-leukemia (GVL) effect following allogenic stem cell transplantation as well as donor lymphocyte infusions indicate that T lymphocytes can control and eliminate AML cells. Leukemia-specific antigenic peptides have been characterized (proteinase-3 and Wilms tumor 1 protein) and serve as targets for peptide-based vaccine trials in AML. Allogenic NK cells have anti-leukemic activity as shown by killer cell inhibitory receptor (KIR)-mismatched haplo-identical stem cell transplantation. Less is known regarding the role of gd T cells in the control of AML. Recently the reconstitution of Vd1 T lymphocytes post transplantation has been shown to correlate with a better prognosis. In the present study, we have analyzed gd T cells in patients with AML and in a mouse model of human AML and focused on (Vg9) Vd2 T cells, the main subset of circulating gd T cells with anti-neoplastic activity. Human Vg9Vd2 T lymphocytes can be activated by nonpeptidic antigens such as the mevalonate pathway-derived isopentenyl pyrophosphate or synthetic phosphoantigen such as bromohydrin pyrophosphate (BrHPP). This population may be suitable for the adoptive immunotherapy of acute myeloid leukemia (AML). However little is known about the frequency, the function and the mechanisms underlying Vg9Vd2 T-cell recognition of AML. We have focused this study on AMoL which are targets of NK and ab T cells. OBJECTIVE OF THE STUDY to describe Vg9Vd2 T cells in patients with AML and investigate their ability to induce an effective cytotoxic response against autologous AML blast in vitro and in vivo. EXPERIMENTAL PROCEDURe: We compared the phenotype and the absolute circulating Vg9Vd2 T cell levels in the blood and the bone marrow (BM) in 12 patients with AMoL (FAB AML-M4 and -M5) and 12 healthy volunteers (HV) using multi parametric flow cytometry. All patients and volunteers gave written informed consent. Vg9Vd2 T cells of AML patient were expanded ex vivo using BrHPP or Zoledronic acid plus IL2. The functions of expanded Vg9Vd2 T cells were assessed in vitro by their cytotoxicity against leukemic blasts (CD107a staining, 51Cr assay) and in vivo in immunodeficient mice transplanted with human AML cell line (U937). In these experiments, the ability of adoptively transferred Vg9Vd2 T cells to migrate into BM and improve mice survival was assessed after i.v. infusion of U937 cells into healthy female NOD-SCID, common _-chain knockout mice (NOG mice). Mice were then treated twice i.v. with 40.106 Vg9Vd2 T cells. RESULTs: Vg9Vd2 T lymphocytes are present in the blood as well as BM of AMoL patients at a lower frequency as compared to HV (median 2.07/μl vs 34/μL respectively P<0.001). Vg9Vd2 T lymphocytes from AML patients are endowed with in vitro proliferation in response to BrHPP or Zoledronic acid plus IL2 but lower than HV (fold increase median 33 versus 69, P=0.051). Expanded Vg9Vd2 express activation markers (CD69 and CCR5) and exhibit an effector/memory phenotype (CD45RA- CD27-). Their lytic potential toward autologous AML blast was equivalent to those of HV by 51Cr experiments and CD107a staining and involves the perforin-granzyme pathway. Their activity depends on both TCRVd2 and DNAX accessory molecule-1 (DNAM-1) as demonstrated by antibody blockade. In vivo data show that, upon sacrifice, Vg9Vd2 were detected in BM, spleen and blood of mice. Preliminary Kaplan-Meier analysis of pooled cohorts of Vg9Vd2-treated and untreated mice reveals that mice receiving Vg9Vd2 T cells displayed superior survival compared with untreated controls (P=0.0047). CONCLUSIOn: Altogether, our data indicate that Vg9Vd2 T cells are decreased in AML patients and have a more limited expansion potential. However, they are able to kill autologous AML blast upon stimulation in a TCRVd2 as well as the DNAM-1 receptor dependent manner. These results provide a rationale for the clinical evaluation of adoptive transfer of ex vivo expanded allogenic Vg9Vd2T cells or direct activation of Vg9Vd2T cells with IL2 + phosphoantigens in patients with AML. Disclosures: No relevant conflicts of interest to declare.

Preclinical Evidence That Trametinib Enhances the Response to Tyrosine Kinase Inhibitors in Acute Myeloid Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1581-1581
Author(s):  
Joaquín Martínez-López ◽  
María Linares ◽  
Maria Luz Morales ◽  
Alicia Arenas ◽  
Ivana Zagorac ◽  
...  
Keyword(s):  
Overall Survival ◽  
Acute Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Ex Vivo ◽  
Tki Treatment ◽  
Acute Myeloid

Abstract Acute Myeloid Leukemia (AML) is the most common type of acute leukemia in adults and the second in children. The overall survival is less than 35% and 60% for adults and children respectively. Activating mutations of FLT3 are now recognized as the most common molecular abnormality in this disease, and the poor prognosis of patients harboring these mutations renders FLT3 an obvious target of therapy. Although different tyrosine kinase inhibitors (TKI) have been used for this purpose, the ability of these drugs to extend progression-free and overall survival in this patient population is limited by drug resistance. This strategy could be improved by rationally combining TKIs with other agents. In this work, we have explored by phosphoproteomics the alternative pathways activated after TKI treatment in vitro and ex vivo. The phosphoproteome profile of the bone marrow from a FLT3-AML patient before and after TKI treatment, studied by LC-MSMS after IMAC enrichment, suggested the activation of Ras-Raf-MEK-ERK1/2 pathway as a possible mechanism for TKI resistance, which could be avoided by dual inhibition using the MEK inhibitor trametinib. Therefore, we characterized the effect of trametinib in combination with the TIK pazopanib and sorafenib by the in vitro cell viability assay using WST8in the FLT3-ITD AML cell line MOLM13. As it is presented in figure 1a, trametinib showed an IC50 value in the low-nanomolar range (5.4 nM) and this MEKI produced a strong synergy (0.5<Ci) with the two TKI tested (figure1b). Moreover, when we analized the activation of the three main pathways downstream the FLT3 receptor by western blot (figure 1c), we observed that the combination of trametinib with both pazopanib and sorafenib showed a complete inhibition of phospho-ERK1/2 compared to the DMSO control (P≤0.0001). Interestingly, we also observed some differences between the two combinations: while trametinib in combination with sorafenib inhibited STAT5 phosphorilation (P≤0.05), the MEKI combination with pazopanib produced a significant decrease on phospho-AKT levels (P≤0.01). In conclusion, we provide preclinical evidence that combining a TKI, such as sorafenib or pazopanib, with a MEKI, such as trametinib, is a rational and efficacious treatment regimen for AML. As trametinib has previously shown good results when combined with pazopanib in clinical trials for other kind of tumors, we expect similar results in AML. On the other hand, trametinib+sorafenib could offer an optimal combination in those patients with elevated levels of phospho-STAT5, as it has been described for patients which present FLT3-ITD mutations. M.L. holds a postdoctoral Fellowship of the Spanish Ministry of Economy and Competitiveness (FPDI-2013-16409) y ML.M. holds a Fellowship of the Spanish Ministry of Education, Culture and Sport (REF-91442). Disclosures Martínez-López: Novartis: Honoraria, Speakers Bureau.

scholarly journals Functional Genetic Variants in ATG10 Are Associated with Acute Myeloid Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1344
Author(s):  
Isabel Castro ◽  
Belém Sampaio-Marques ◽  
Anabela C. Areias ◽  
Hugo Sousa ◽  
Ângela Fernandes ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Genetic Variants ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
Metabolic Reprogramming ◽  
Peripheral Blood Mononuclear ◽  
Increased Risk ◽  
And Gender ◽  
Acute Myeloid

Acute myeloid leukemia (AML) is the most common acute leukemia, characterized by a heterogeneous genetic landscape contributing, among others, to the occurrence of metabolic reprogramming. Autophagy, a key player on metabolism, plays an essential role in AML. Here, we examined the association of three potentially functional genetic polymorphisms in the ATG10 gene, central for the autophagosome formation. We screened a multicenter cohort involving 309 AML patients and 356 healthy subjects for three ATG10 SNPs: rs1864182T>G, rs1864183C>T and rs3734114T>C. The functional consequences of the ATG10 SNPs in its canonical function were investigated in vitro using peripheral blood mononuclear cells from a cohort of 46 healthy individuals. Logistic regression analysis adjusted for age and gender revealed that patients carrying the ATG10rs1864182G allele showed a significantly decreased risk of developing AML (OR [odds ratio] = 0.58, p = 0.001), whereas patients carrying the homozygous ATG10rs3734114C allele had a significantly increased risk of developing AML (OR = 2.70, p = 0.004). Functional analysis showed that individuals carrying the ATG10rs1864182G allele had decreased autophagy when compared to homozygous major allele carriers. Our results uncover the potential of screening for ATG10 genetic variants in AML prevention strategies, in particular for subjects carrying other AML risk factors such as elderly individuals with clonal hematopoiesis of indeterminate potential.

scholarly journals In Vitro Approach for the Identification of Exceptional Responders in Acute Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2212-2212
Author(s):  
Eric O'Brien ◽  
Brett VanCauwenbergh ◽  
Luke Byerly ◽  
Alexander Merk ◽  
Mayur Sarangdhar ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Drug Sensitivity ◽  
Cell Function ◽  
Ex Vivo ◽  
In Vitro Drug Screening ◽  
Pediatric Aml ◽  
Acute Myeloid

Abstract Each case of Acute Myeloid Leukemia (AML) represents a unique ecosystem. Despite recent advances in our understanding of the genetic landscape of AML, this information remains insufficient to accurately match patients with targeted therapies. While pediatric and adult AML share phenotypic similarities, pediatric AML represents a genetically distinct disease from adult AML, and will benefit from independent genomic studies and novel therapeutic strategies. Real-time ex-vivo functional screening can identify mechanisms underpinning drug response and diversity between tumors, aiding in patient stratification. We established an in vitro drug screening system that incorporates cytokine signaling to better model inflammation, stem cell function, and niche derived support of leukemic blasts. This approach provided insight into variability between patients who currently would be placed on the same therapeutic regimen. Samples were acquired from 12 pediatric AML patients, after informed consent was obtained. Samples were enriched for blasts, and cultured in the presence of SCF, TPO, FLT3-L, IL-3, and IL-6 (KTF36). A panel of 38 drugs was selected from a larger screen of 1839 compounds done on commercially available hematological malignancy cell lines. Drugs included standard chemotherapy agents used in AML and drugs currently under clinical development. Cells were exposed to drugs for 72hrs. An MTS assay was performed and results reported as % of viable cells remaining, after normalization to vehicle control wells. Targeted DNA NGS sequencing of 406 genes, 31 introns, and RNA sequencing of 265 genes was performed for genetic characterization. In vitro drug screening revealed variations in drug sensitivity between samples and revealed time ex-vivo and cytokine milieu to be important factors affecting response of the same cells to the same drugs. Engraftment of long term cultures into immunodeficient mice produced aggressive disease in all cases, indicating robust support of stem cell function via addition of KTF36 cytokines. When possible, clinical response to therapy was compared with in vitro response to the same drugs. This screening approach highlighted well established agents that showed significant activity in highly refractory disease providing rationale for further clinical trial development. An unsupervised clustering showed drug sensitivity primarily correlated with the presence of MLL-X fusion, NRAS/KRAS and PTPN11 alterations. Linear Regression with interaction effects showed drug sensitivity/resistance to be highly selective for single/signature of specific molecular alterations. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Targeting and Depletion of Acute Myeloid Leukemia Blasts By MEN1112, a Novel Humanized Defucosylated Monoclonal Antibodies with Specificity for Bst1/CD157 Antigen

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2235-2235
Author(s):  
Adriano Venditti ◽  
Francesco Buccisano ◽  
Luca Maurillo ◽  
Maria Ilaria Del Principe ◽  
Andrea Coppola ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Ex Vivo ◽  
Antigen Expression ◽  
Distinct Pattern ◽  
Healthy Donors ◽  
Acute Myeloid

Abstract MEN1112 is a new humanized, defucosylated, monoclonal antibody (mAb) with high specific affinity for Bst1/CD157 antigen. Bst1/CD157 antigen expression on blood cells of acute myeloid leukemia (AML) patients and healthy donors was investigated by flow cytometry using a PE-labeled MEN1112 mAb. Twenty three patients affected with AML have been tested, 18 at diagnosis, 4 at relapse, 1 resistant. In 16 out of 23 patients both bone marrow (BM) and peripheral blood (PB) specimens were evaluated. PB and BM samples from healthy donors (N=2) were also assessed. In healthy donors and AML patients, PB and BM lymphocytes were Bst1/CD157 antigen negative whereas monocytes and neutrophils showed a distinct pattern of MEN1112 mean fluorescence intensity (MIF), with monocytes having the brightest expression. In the stem cell compartment, an intermediate level of MFI was observed (p<0.001). Prevalence of expression of the antigen on patients’ samples was over 90%. On AML blast cells from each single patient, MEN1112 expression was heterogeneous; indeed the antigen was expressed on 50%±29% and 47% ±39% of blasts in BM and PB, respectively. The anti-leukemia activity of MEN1112 on AML cell lines was tested, in vitro, by a flow cytometry-based cell depletion assay in the presence of lymphokine activated immune effector cells: a strong depletion of leukemia cells was demonstrated suggesting that MEN1112 might exert anti-leukemia activity through antibody dependent cell-mediated cytotoxicty (ADCC). The activity of MEN1112 was also tested ex vivo on whole PB showing that the antibody was able to deplete AML blasts in 9 out of 23 patients (47.4 %) with a percentage of AML blast depletion ranging between 4.3 - 66 %. In whole BM from 2 out of 11 evaluable patients MEN1112 induced 68% and 23% of AML blast depletion. Bst1/CD157 shedding assessment showed that, in the sera from AML samples, the concentration of Bst1/CD157 antigen was comparable to that measured in healthy donors. Moreover, since Fcγ receptor (CD16) genotype might be a factor contributing to the antitumor activity of the antibody, the polymorphism CD16-158Phe/Val was analyzed. Five out of 19 samples were homozygous for CD16-158 Phe; 5 were homozygous for CD16- 158 Val and 9 were heterozygous for CD16-158. MEN1112-induced blast depletion was observed for each genotype. Moreover, in an attempt to identify the determinant of MEN1112 activity, % in PB of blast (antigen positive), NK cells or residual normal cells were evaluated. Altogether, these results are promising suggesting the potential for an ADCC-mediated MEN1112 antileukemic effect and they support the clinical development of MEN1112. Disclosures Venditti: Menarini Ricerche SpA: Research Funding. Buccisano:Menarini Ricerche SpA: Research Funding. Del Principe:Menarini Ricerche SpA: Research Funding. Coppola:Menarini Ricerche SpA: Research Funding. Palomba:Menarini Ricerche SpA: Research Funding. Aureli:Menarini Ricerche SpA: Research Funding. Arriga:Menarini Ricerche SpA: Research Funding. Bellarosa:Menarini Spa: Employment. Bressan:Menarini Ricerche SpA: Employment. Manzini:Menarini Ricerche SpA: Employment. Simonelli:Menarini Ricerche SpA: Employment. Binaschi:Menarini Ricerche SpA: Employment. Amadori:Menarini Ricerche SpA: Research Funding. Sconocchia:Menarini Ricerche SpA: Research Funding.

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