Lymphocyte Cytosolic Protein 1 (LCP1) Is a Membrane Associated Molecular Target in Chronic Lymphocytic Leukemia and Is Activated in Microenvironment Signaling

Abstract 3866

Chronic lymphocytic leukemia (CLL) is an incurable accumulation of malignant B-lymphocytes in peripheral circulation, which home to supportive leukemic microenvironments within the bone marrow, lymph nodes, and spleen. Membrane associated antigens are critical to the pathogenesis of CLL since they facilitate leukemic cell signaling, microenvironment homing, proliferation, and survival. Targeting CLL surface molecules and associated signaling patterns is a current focus of CLL therapeutic development with antibodies and vaccine-based approaches. Rituximab, ofatumumab, and alemtuzumab are widely used antibody-based therapeutics that are proven to be effective at targeting CLL membrane associated antigens. However, despite enhanced patient survival, targeting of non-obligate antigens such as CD20 ultimately leads to antibody resistance. In addition, the expression of CD52 across multiple healthy immune cells often produces unacceptable off-target toxicity. Targeting immunosurveilance antigens may provide a key to overcome these obstacles. A number of relevant tumor antigens such as p53, EGFR, WT1, BCR-Abl and CLL-specific ROR1 elicit robust autoimmune responses in cancer patients due to high levels of ectopic expression, high relative immunogenicity and obligate oncogenic signaling that forces expression despite autoimmune recognition. In many cases, autoantibody signatures were realized long after identification of the oncologic target antigens; however, the convergence of these two datasets implies that such signatures could be utilized to identify novel targets. To further explore this possibility, we interrogated anti-tumor humoral reactivity specifically directed against autologous CLL membrane antigens. Immunoreactive leads were identified by mass spectrometry. Potential membrane antigens were further confirmed using immunoblot and ELISA based techniques. Our analysis revealed Lymphocyte Cytosolic Protein 1 (LCP1), a membrane associated lymphocyte-specific target that is constitutively expressed on CLL and ectopically expressed in cancers of various histological subtypes. Subsequent confirmatory assays unveiled high frequency, robust, LCP1-specific IgG autoimmune responses in CLL patients despite a profound absence of reactivity to common viral and vaccine antigens, implying continued antigenic stimulation in an immunosuppressed host. LCP1 plays a critical role in B-cell biology by cross linking f-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for CLL-critical signaling pathways such as PKC. In healthy B-cells phosphorylation of LCP1 at serine 5 is a key step in responding to CXCL12, a powerful lymph node and stromal cell chemokine. To further explore the role of LCP1 in CLL microenvironment recall signaling, we developed stable LCP1 knockdown and Ser-5 phosphomimetic CLL cell lines. Transwell assays confirmed that LCP1 activity contributed to the migration of CLL cells towards CXCL12. Our data reveals that LCP1 is a novel membrane associated target antigen in CLL with evidence of differential immune response as compared to common vaccine antigens. We also demonstrate that LCP1 plays a role in CLL microenvironment signaling. Future studies will focus on targeting specific moieties within LCP1 which are critical for its oncogenic signature.