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3392 FLT3-ITD Driven CMTM6 Expression Contributes to Immune Escape of Acute Myeloid Leukemia

Program: Oral and Poster Abstracts
Session: 701. Experimental Transplantation: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Acute Myeloid Malignancies, AML, Translational Research, Diseases, Immune mechanism, Treatment Considerations, Biological therapies, Immunotherapy, Immunology, Myeloid Malignancies, Biological Processes, Transplantation (Allogeneic and Autologous)
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Melissa Zwick1,2*, Bastian Zinkel1,2*, Corinna Spohr3*, Tamina Rückert1*, Sebastian Halbach3,4*, Khalid Shoumariyeh, MD1,4*, Lukas M. Braun1,2*, Annika Nelde5,6*, Melanie Maerklin6,7*, Samuel J. Holzmayer6,7*, Sandra Duquesne1*, Alexandra Emilia Schlaak8*, Patricia Otto-Mora8*, Bertram Bengsch8,9,10,11*, Marcel Schiff1*, Sandra Kissel1*, Marie Follo1*, Heidi Altmann12*, Désirée Kunadt12*, Anna Lena Illert1,4,13,14*, Juliane S. Walz, MD5,6,7,15*, Gerd Walz9,16,17*, Justus Duyster, MD1,18*, Johannes Schetelig, MD, MSc11,12, Tilman Brummer, PhD3,4,9,19*, Robert Zeiser, MD1,4,9,17 and Natalie Koehler1,17*

1Department of Medicine I, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
2Faculty of Biology, University of Freiburg, Freiburg, Germany
3Institute of Molecular Medicine and Cell Research (IMMZ), Zentrum für Biochemie und Molekulare Zellforschung (ZBMZ), Faculty of Medicine, University of Freiburg, Freiburg, Germany
4German Cancer Consortium (DKTK), Partner Site Freiburg and German Cancer Research Center (DKFZ), Heidelberg, Germany
5Department of Peptide-based Immunotherapy, Institute of Immunology, University and University Hospital Tübingen, Tübingen, Germany
6Cluster of Excellence iFIT (EXC2180) "Image-Guided and Functionally Instructed Tumor Therapies", University of Tübingen, Tübingen, Germany
7Clinical Collaboration Unit Translational Immunology, Department of Internal Medicine, University Hospital Tübingen, Tübingen, Germany
8Department of Internal Medicine II (Gastroenterology, Hepatology, Endocrinology and Infectious Diseases), Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
9BIOSS Center for Biological Signalling Studies, University of Freiburg, Freiburg, Germany
10German Cancer Consortium (DKTK), Heidelberg, Germany
11Clinical Trials Unit, DKMS gGmbH, Dresden, Germany
12Department of Internal Medicine I, University Hospital Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany
13Department of Medicine III, Klinikum Rechts der Isar, Faculty of Medicine, Technical University of Munich, Munich, Germany
14TranslaTUM, Center for Translational Cancer Research, Technical University of Munich, Munich, Germany
15German Cancer Consortium (DKTK), Partner Site Tübingen and German Cancer Research Center (DKFZ), Tübingen, Germany
16Renal Division, Medical Center – University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany
17CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany
18German Cancer Consortium (DKTK), Partner site Freiburg; and German Cancer Research Center (DKFZ), Heidelberg, Germany
19Comprehensive Cancer Center Freiburg (CCCF), Faculty of Medicine and Medical Center, University of Freiburg, Freiburg, Germany

Introduction:
Acute myeloid leukemia (AML) is a highly diverse blood cancer caused by genetic mutations, including FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD), which occurs in about 25% of AML cases and is linked to a high leukemic burden and poor prognosis. While allogeneic hematopoietic cell transplantation (allo-HCT) can be a curative treatment for high-risk AML, post-transplant relapse remains a common and challenging issue, frequently leading to death. Relapse after allo-HCT often results from the loss of the graft-versus-leukemia (GVL) effect due to immune escape mechanisms like programmed death-ligand 1 (PD-L1) expression. CKLF-like MARVEL transmembrane domain containing member 6 (CMTM6) was identified to bind and stabilize PD-L1, thereby maintaining its expression at the cell surface in solid tumor cells. In this study, we found that oncogenic FLT3-ITD drives CMTM6 and PD-L1 protein expression in leukemia cells, contributing to immune escape of AML cells.

Methods:
For in vitro studies, 32D and WEHI-3B cell lines transduced with pMig vectors carrying different oncogenic mutations and the human MV4-11 cell line were used to assess the effect of oncogenes on the PD-L1/CMTM6 axis via flow cytometry, qRT-PCR, co-immunoprecipitation (Co-IP) and immunofluorescence microscopy. FLT3 kinase inhibition assays were performed using the FLT3 inhibitors tandutinib, crenolanib, and quizartinib.
To study in vivo effects, an allo-HCT mouse model with wild type (WT) or Cmtm6-deficient C57BL/6N bone marrow (BM) transduced with FLT3-ITD and BALB/c donor BM / T cells was analyzed for survival and T cell activation by flow cytometry and CyTOF.
Additionally, a Rag2-/-γc-/- xenograft mouse model with pTRIPZ inducible CMTM6-knockdown or WT MV4-11 cells and human T cells was used to study the effect of CMTM6 on disease progression. Clinical relevance was assessed using peripheral blood samples from FLT3-ITD+ AML patients (n=23) and bone marrow aspirates from AML patients before and after relapse (n=17), provided by the Study Alliance Leukemia Biobank.

Results:
Transduction of murine AML cell lines and primary BM cells with different oncogenes revealed that FLT3-ITD mutation enhanced protein expression of CMTM6 and PD-L1, which was reversible when treating cells with FLT3 inhibitors.

Mechanistically, we found FLT3 kinase co-localized with CMTM6 in the plasma membrane and endoplasmic reticulum in AML cell lines. Co-IP of FLT3 and CMTM6 in FLT3-ITD+ AML cells confirmed their interaction at the protein level, while FLT3-ITD did not increase Cmtm6 mRNA expression.

In the FLT3-ITD+ leukemia allo-HCT mouse model, Cmtm6-deficient AML mice showed significantly prolonged survival (p=0.005) and reduced leukemia burden (p=0.002) compared to WT AML mice. FLT3-ITD+ Cmtm6-deficient AML cells also showed decreased PD-L1 levels in peripheral blood and spleen ultimately leading to enhanced T cell activation with a significantly higher abundance of CD4 and CD8 effector T cells and reduced naïve T cell numbers in the spleen. In addition, effector T cells showed increased CD69 and IFNγ expression.

In a second mouse model, knockdown of CMTM6 in MV4-11 cells reduced CMTM6/PD-L1 surface expression causing improved AML rejection (p=0.003) and increased T cell activation measured by CD69 expression on CD4 T cells. Similarly, tandutinib treatment reduced leukemia burden as well as CMTM6 protein levels in Rag2-/-γc-/- mice transplanted with WT MV4-11 cells.

In CD34+ blasts derived from PBMCs and BM aspirates of 2 independent AML patient cohorts, we found elevated CMTM6 protein expression in FLT3-ITD+ patients, which was associated with reduced T cell effector function and activation.

Conclusions:
We found that oncogenic FLT3-ITD increased CMTM6 and PD-L1 expression in AML cells, while FLT3 inhibition reduced their expression. Transplantation of Cmtm6-deficient FLT3-ITD+ leukemia cells improved survival, reduced leukemia burden and enhanced T cell activation compared to Cmtm6-proficient FLT3-ITD+ leukemia cells in vivo. We confirmed enhanced CMTM6 expression on leukemia cells in 2 independent cohorts of FLT3-ITD+ AML patients. Taken together, our study identifies a new mechanism of oncogene-induced stabilization of CMTM6 in leukemia cells, resulting in tumor immune escape. Inhibiting the CMTM6/PD-L1 axis could enhance the GVL effect post allo-HCT in patients with FLT3-ITD+ AML.

Disclosures: Shoumariyeh: Blueprint: Honoraria. Walz: ViferaXS GmbH: Current equity holder in private company; Swarm Oncology: Consultancy; Organoid Science: Consultancy. Schetelig: Janssen: Consultancy, Honoraria; MSD: Consultancy; Novartis: Honoraria; Eurocept: Honoraria; Astellas: Honoraria; Medac: Honoraria; AstraZeneca: Consultancy, Honoraria. Brummer: Pierre Fabre: Honoraria. Zeiser: Medac: Honoraria; Sanofi: Honoraria; Neovii: Consultancy; Novartis: Consultancy, Honoraria; Ironwood Pharmaceuticals, Inc.: Consultancy; Incyte: Consultancy, Honoraria; Mallinkrodt: Consultancy, Honoraria.

*signifies non-member of ASH