Background: Developing early interventions for diffuse large B cell lymphoma (DLBCL) and B cell acute lymphoblastic leukemia (B-ALL) requires an understanding of the molecular mechanisms underlying the initiation, establishment, and sustenance of these high-risk B cell malignancies. B cell cancers are initiated 3-8-fold more frequently in patients suffering from refractory autoimmune B-lymphoproliferative disorders such as systemic lupus erythematosus (SLE), making SLE a relevant disease model to study initiation of B cell cancers. The hormone prolactin (PRL) exacerbates the symptoms of SLE, enhances lymphoid cell survival, and promotes the expression of MYC and BCL2 protooncogenes in lymphocytes. PRL signals via prolactin receptors (PRLRs). PRLRs have long (LF), intermediate (IF, only in humans) and short (SF) isoforms generated by alternative splicing. Increased expression of the LF/IF relative to the SF PRLR on cells leads to cell proliferation and survival, whereas increased expression of SF relative to LF/IF inhibits proliferation and induces apoptosis.
Aims: Whether PRL drives initiation, establishment, and sustenance of B cell malignancies was unknown. We hypothesized that PRL, by signaling specifically through the pro-proliferative, anti-apoptotic LF/IFPRLR drives the three stages of B cell malignancy development, namely, initiation (Stage 1), establishment (Stage 2), and sustenance (Stage 3). To test this, we measured the effects of LF/IFPRLR knockdown on B cells in vivo in transgenic mouse models of SLE (Stage 1/ Aim 1), DLBCL (Stage 2/ Aim 2), and in vitro in malignant human B cells (Stage 3/ Aim 3).
Methods: To knockdown LFPRLR in mice and LF/IFPRLR in human cells without suppressing expression of the SFs, we used a patented splice-modulating DNA morpholino oligomer (SMO), termed LFPRLR SMO, that prevents the splicing event needed to produce LF in mice and LF/IF in humans.
Results: LFPRLR knockdown reduced splenic B cell numbers in SLE-prone mice. Consistent with reduced B cell numbers, splenic B cell proliferation and subsequent generation of antibody-producing plasma cells was significantly lowered after LFPRLR knockdown. LFPRLR SMO-treated SLE-prone mice had reduced frequencies of deleterious splenic B cells with long immunoglobulin heavy chain (IGH) complementary determining region 3 (CDR3) >20 amino acids and B cells with non-functional IGH that are prone to malignant transformation. LFPRLR knockdown in SLE-prone mice also significantly reduced the expression of the activation induced cytidine deaminase (AID) enzyme, known to induce malignant transformation of B cells. Therefore, we infer that PRL-LFPRLR signaling exacerbates SLE B cell pathology and promotes malignant transformation of SLE B cells (Stage 1 of B cell malignancy development). In DLBCL-prone mice carrying pre-malignant B cell clones, LFPRLR knockdown strikingly reduced splenic B cell counts and expression of BCL2 in B cells. Thus, PRL-LFPRLR signaling drives stage 2 of B cell malignancy development, i.e., establishment of overt B cell neoplasms from pre-malignant B cells. Finally, in overt human DLBCL and B-ALL (Stage 3), we found that knockdown of LF/IFPRLR reduced the expression of MYC and BCL2 and impaired viability of malignant B cells.
Summary/Conclusion: LF/IFPRLR is a safe and isoform-specific prophylactic and therapeutic target to alleviate pathology of B cell subsets and concurrently lower the incidence of B cell malignancies in SLE patients (Stage 1), reduce the risk of B cell malignancies in people with pre-malignant and indolent B cell clones (Stage 2), and treat overt B cell neoplasms (Stage 3).