Identification of Cmtm Family Proteins As Tumor Suppressor and Membrane Regulator in B Cell Precursor Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is characterized by the expansion of immature hematopoietic cells in the bone marrow and blood. It is the most common childhood malignancy and is one of the major causes of death in children. Treatment improvements have increased the cure rate to more than 80% for children and about 40% for adults. However, current chemotherapy causes acute and long-term toxicity. Hence, there is a compelling need to understand the development of ALL and identify key players that may be used in targeted therapy. The B-cell receptor (BCR) and its precursor, pre-BCR, control B cell development, which is arrested in ALL. This blockade occurs at the first quality control checkpoint of B-cell development; the pre BCR checkpoint. To enhance our understanding of B-cell precursor (BCP)-ALL specific membrane associated signaling, we have investigated the functions of the recently discovered Chemokine factor like Marvel like Trans Membrane proteins (CMTM) that interact with BCR in pre-BCR checkpoint.

We have identified heterozygous focal deletions of CMTM family genes, specifically CMTM6, 7 and 8 in BCP-ALL, including the subtype with intrachromosomal amplification of chromosome 21 (iAMP21). Similar focal deletions were found in iAMP21 xenograft models after serial passage, indicating their possible link to survival advantage. Although CMTM family proteins were first described in 2003, little is known about their physiological functions. The loss of a small gene cluster at chromosome 3p22, including CMTM6 and 7 has been reported in several cancers, including esophageal squamous cell, nasopharyngeal and lung carcinomas, indicating their potential roles as tumor suppressor genes. Among the CMTM family members, CMTM3 and 7 were initially identified as interacting partners of B-cell receptors. To characterize the CMTM mediated macromolecular assemblage in BCP-ALL, immunoprecipitation (IP) studies were performed using CMTM7 antibody. Initially, the expression of CMTM7 and the sensitivity of the CMTM7 antibodies were tested using various BCP-ALL cell lines. Due to their positive expression levels, the pre-B697 and NALM6 cell lines were selected for the IP studies. When the CMTM7-mediated membrane protein complex was isolated using CMTM7 antibody, we determined that the well-established tumor suppressor, B-cell linker (BLNK), interacted with CMTM7 in pre-B697 and NALM6. CMTM7-interaction partners are being verified by mass spectrometry. Next, to identify possible physiological functions, we performed a phylogenetic analysis and discovered that the CMTM family genes were homologous to myelin and lymphocyte (MAL) proteins, tricellulins, plasmolipins and occludin families, which comprise the tetra-spanin trans-membrane domain known as MARVEL (MAL and related protein for vesicle trafficking and membrane linking). These proteins are associated with cell communication and intracellular transport. To investigate the molecular mechanism of action of CMTM6, 7 and 8 in BCP-ALL and B cell development, we cloned, expressed and purified all three members and preliminary functional studies in-vitro indicated that they formed oligomers. Taken together, these data identify a critical membrane regulator, CMTM7, which may function as a tumor suppressor, communicating signals from membrane to cytosolic components through BLNK signaling in BCP-ALL.