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2730 CBL Loss or Mutations Reduces MHCII Expression through Downregulating ERG in Myeloid Malignancies

Program: Oral and Poster Abstracts
Session: 602. Myeloid Oncogenesis: Basic: Poster II
Hematology Disease Topics & Pathways:
Adult, Fundamental Science, Research, Acute Myeloid Malignancies, AML, MPN, Genomics, Bioinformatics, Chronic Myeloid Malignancies, CMML, Diseases, Immune mechanism, Immunotherapy, Treatment Considerations, Biological therapies, Immunology, Myeloid Malignancies, Biological Processes, Gene editing, Technology and Procedures, Molecular biology, Human, Study Population, Profiling, Animal model, Transplantation (Allogeneic and Autologous)
Sunday, December 8, 2024, 6:00 PM-8:00 PM

Rouxing Wang, PhD1,2, Mohammed Alhusayan3*, Bowen Xing1,2*, Emma Hedgepeth4*, Yan Liu, PhD5, Hamid Band, MD, PhD6*, Laurence Eisenlohr4*, Junwei Shi, PhD3*, Irfan Asangani3* and Wei Tong, PhD2,7

1Division of Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
2Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
3Department of Cancer Biology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA
4Department of Pathology and Laboratoy Medicine, University of Pennsylvania, The Children's Hospital of Philadelphia, Philadelphia, PA
5Northwestern University, Chicago, IL
6Nebraska Medicine, University of Nebraska Medical Center, Omaha, NE
7Children's Hospital of Philadelphia, Philadelphia, PA

Reduced expression of major histocompatibility complex class II (MHCII) has been associated with immune escape and relapse of acute myeloid leukemia (AML) after allogeneic hematopoietic cell transplantation (Christopher, NEJM, 2018; Gambacorta, Cancer Discov, 2022). Normal hematopoietic stem and progenitor cells (HSPCs), the origin of hematopoietic malignancies, were shown to constitutively present self-antigen via MHCII for immune surveillance (Hernandez, CSC, 2022). Regulation of MHCII expression in AML and HSPCs is highly clinically relevant but the mechanism is not completely known.

Loss-of-function mutations in CBL (Casitas B-lineage Lymphoma), an E3 ubiquitin ligase, are found in a wide range of myeloid malignancies most frequently occurring in chronic and juvenile myelomonocytic leukemia (CMML and JMML). CBL mutations are associated with poor prognosis; however, the therapeutic targets are poorly established. Through quantitative proteomics studies, we discovered that double deletion of CBL and CBLB (CBL-DKO) or overexpression of the E3-mutant CBL C381A in human leukemia cell lines, resulted in dramatically decreased expression of most if not all MHCII components. RNAseq studies revealed that MHCII gene expressions were downregulated in CBL-DKO and CBL mutant cells compared with control or wildtype CBL expressing cells. This correlates with the downregulation of MHCII master transactivator, CIITA. ChIPseq analysis revealed decreased active epigenetic marks (H3K27Ace, H3K4me3) at the MHCII locus and the CIITA locus in CBL-DKO and mutant CBL cells. Interferon-γ, which has long been known to upregulate MHCII in a variety of cells, rapidly induced MHCII protein expression in CBL-DKO cells. The reversibility of MHCII downregulation in these cells suggests that this phenomenon is mediated by an epigenetic mechanism. Importantly, exogenous CIITA expression also promoted MHCII expression in CBL-DKO cells.

To study the mechanism of decreased CIITA/MHCII expression in CBL-DKO and CBL mutant cells, we performed CRISPR-Cas9 screens targeting functional domains of transcription factors and epigenetic enzymes. This identified ERG as one of the major positive regulators of MHCII. Importantly, ERG expression is downregulated in CBL-DKO and CBL mutant cells. We next performed epigenetic profiling of active and suppressive marks combined with published ChIPseq datasets that examined ERG genome occupancy in human HSPCs, AML, and B lymphoma cell lines (Thoms, Blood 2021; Kodgule, Blood Cancer Discov 2023; Subramanian, Blood 2023). We found that ERG is bound to the CIITA gene region enriched for H3K27Ace in AML cell lines and human HSPCs, at which location distinct from that in B lymphoma cells. Specifically, ERG predominantly binds to the enhancer in the first intron of CIITA downstream of promoter pIII/pIV in AML and HSPCs, while it binds to an enhancer at the 3’ end of the gene in B cells. Importantly, mutating or deleting these ERG binding motifs within this candidate enhancer in AML cells markedly reduced the MHCII level, implicating a critical role for ERG in maintaining CIITA transcription and thereby MHCII expression.

To confirm the relationship between CBL and MHCII expression in vivo, we utilized tamoxifen-induced Cbl/Cblb conditional knockout mice (Cblf/f;Cblbf/f;CreERT), which develop an aggressive CMML-like disease (Lv, G&D, 2017). Corroborating with our cell line data, Cbl/Cblb deficient HSPCs, but not antigen-presenting cells (B cells), displayed significantly reduced MHCII levels. Importantly, the capacity to activate CD4+ T cells was abrogated in Cbl/Cblb deficient HSPCs but not B cells.

Lastly, we examined MHCII levels in primary AML samples. CBL-mutant AMLs showed decreased MHCII levels compared with AMLs without CBL mutation. In addition, we reported one AML case, where AML blasts gained a new CBL mutation upon relapse after allogeneic HSCT. Examining additional AML samples is in progress.

Loss of MHCII expression represents a frequent mechanism of post-transplantation relapse of AML. Here we discover that CBL loss or mutations reduce the expression of MHCII in myeloid malignancies through downregulating HSPC heptad transcription factor ERG. This study links immune escape to specific AML driver mutations in a signaling molecule, providing a new therapeutic target to reinstate a proficient graft-versus-leukemia effect.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH