Targeting CD19-positive lymphomas with the antibody-drug conjugate loncastuximab tesirine: preclinical evidence as single agent and in combination therapy.

Antibody-drug conjugates (ADCs) represent one of the most successful therapeutic approaches introduced in clinical practice in the last few years. Loncastuximab tesirine (ADCT-402) is a CD19 targeting ADC, in which the antibody is conjugated through a protease cleavable dipeptide linker to a pyrrolobenzodiazepine (PBD) dimer warhead (SG3199). Based on the results of a phase 2 study, loncastuximab tesirine was recently approved for adult patients with relapsed/refractory large B-cell lymphoma. We assessed the activity of loncastuximab tesirine using in vitro and in vivo models of lymphomas, correlated its activity with CD19 expression levels, and identified combination partners providing synergy with loncastuximab tesirine. Loncastuximab tesirine was tested across 60 lymphoma cell lines. Loncastuximab tesirine had strong cytotoxic activity in B-cell lymphoma cell lines. The in vitro activity was correlated with CD19 expression level and intrinsic sensitivity of cell lines to the ADC's warhead. Loncastuximab tesirine was more potent than other anti-CD19 ADCs (coltuximab ravtansine, huB4-DGN462), albeit the pattern of activity across cell lines was correlated. Loncastuximab tesirine activity was also largely correlated with cell line sensitivity to R-CHOP. Combinatorial in vitro and in vivo experiments identified the benefit of adding loncastuximab tesirine to other agents, especially BCL2 and PI3K inhibitors. Our data support the further development of loncastuximab tesirine as a single agent and in combination for patients affected by mature B-cell neoplasms. The results also highlight the importance of CD19 expression and the existence of lymphoma populations characterized by resistance to multiple therapies.

PI3Kδ activation, IL6 over-expression, and CD37 loss cause resistance to the targeting of CD37-positive lymphomas with the antibody-drug conjugate naratuximab emtansine.

Purpose: The transmembrane protein CD37 is expressed almost exclusively in lymphoid tissues, with the highest abundance in mature B cells. CD37-directed antibody- and, more recently, cellular-based approaches have shown preclinical and promising early clinical activity. Naratuximab emtansine (Debio 1562, IMGN529) is an antibodydrug conjugate (ADC) that incorporates an anti-CD37 monoclonal antibody conjugated to the maytansinoid DM1 as payload. Naratuximab emtansine has shown activity as a single agent and in combination with the anti-CD20 monoclonal antibody rituximab in B cell lymphoma patients. Experimental Design: We assessed the activity of naratuximab emtansine using in vitro models of lymphomas, correlated its activity with CD37 expression levels, characterized two resistance mechanisms to the ADC, and identified combination partners providing synergy. Results: The anti-tumor activity of naratuximab emtansine was tested in 54 lymphoma cell lines alongside its free payload. The median IC 50 of naratuximab emtansine was 780 pM, and the activity, primarily cytotoxic, was more potent in B than in T cell lymphoma cell lines. In the subgroup of cell lines derived from B cell lymphoma, there was some correlation between sensitivity to DM1 and sensitivity to naratuximab emtansine (r=0.28, P = 0.06). After prolonged exposure to the ADC, one diffuse large B cell lymphoma (DLBCL) cell line developed resistance to the ADC due to the biallelic loss of the CD37 gene. After CD37 loss, we also observed upregulation of IL6 (IL-6) and other transcripts from MYD88/IL6-signaling. Recombinant IL6 led to resistance to naratuximab emtansine, while the anti-IL6 antibody tocilizumab improved the cytotoxic activity of the ADC in CD37-positive cells. In a second model, resistance was sustained by an activating mutation in the PIK3CD gene, associated with increased sensitivity to PI3K δ inhibition and a switch from functional dependence on the anti-apoptotic protein MCL1 to reliance on BCL2. The addition of idelalisib or venetoclax to naratuximab emtansine overcame resistance to the ADC in the resistant derivative while also improving the cytotoxic activity of the ADC in the parental cells. Conclusions: Targeting B cell lymphoma with the CD37 targeting ADC naratuximab emtansine showed vigorous anti-tumor activity as a single agent, which was also observed in models bearing genetic lesions associated with inferior outcomes, such as MYC translocations and TP53 inactivation or resistance to R-CHOP. Resistance DLBCL models identified active combinations of naratuximab emtansine with drugs targeting IL6, PI3K δ , and BCL2. Despite notable progress in recent decades, we still face challenges in achieving a cure for a substantial number of lymphoma patients (1,2). A pertinent example is diffuse large B cell lymphoma (DLBCL), the most prevalent type of lymphoma (3). More than half of DLBCL patients can achieve remission, but around 40% of them experience refractory disease or relapse following an initial positive response (3). Regrettably, the prognosis for many of these cases remains unsatisfactory despite introducing the most recent antibody-based or cellular therapies (3,4), underscoring the importance of innovating new therapeutic strategies and gaining insights into the mechanisms of therapy resistance. CD37 is a transmembrane glycoprotein belonging to the tetraspanin family, primarily expressed on the surface of immune cells, principally in mature B cells but also, at lower levels, in T cells, macrophages/monocytes, granulocytes and dendritic cells (5) (6-8). CD37 plays a crucial role in various immune functions, including B cell activation, proliferation, and signaling, although its precise role still needs to be fully elucidated. CD37 interacts with multiple molecules, including SYK, LYN, CD19, CD22, PI3K δ , PI3K γ , and different integrins, among others (6-8). In mice, the lack of CD37 is paired with reduced T cell-dependent antibody-secreting cells and memory B cells, apparently due to the loss of CD37-mediated clustering of α 4 ß 1 integrins (VLA-4) on germinal center B cells and decreased downstream activation of PI3K/AKT signaling and cell survival (5). Reflecting the expression pattern observed in normal lymphocytes, CD37 exhibits elevated expression in all mature B-cell lymphoid neoplasms, including most lymphoma subtypes, and absence in early progenitor cells or terminally differentiated plasma cells (6,8-14). In DLBCL, CD37 expression has been reported between 40% and 90% of cases across multiple studies performed using different antibodies (10,14-16). CD37-directed antibody- and, more recently, cellular-based approaches have shown preclinical (7,10-14,17-23) and early promising clinical activity (24-32). Among the CD37-targeting agents, naratuximab emtansine (Debio 1562, IMGN529) is an antibody-drug conjugate (ADC) that incorporates the anti-CD37 humanized IgG1 monoclonal antibody K7153A conjugated to the maytansinoid DM1, as payload, via the thioether linker, N-succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (10). Based on the initial in vitro and in vivo evidence of anti-tumor activity in lymphoma and chronic lymphocytic leukemia (CLL) (7,10), naratuximab emtansine entered the clinical evaluation as a single agent. The phase 1 study exploring naratuximab emtansine enrolled 39 patients with relapsed/refractory B cell lymphoma (27). The overall response rate (ORR) was 13% across all patients and 22% in DLBCL patients, including the only observed complete remission (CR) (27). In preliminary results of a phase 2 trial exploring the combination of naratuximab emtansine with the anti-CD20 monoclonal antibody rituximab (18), based on positive preclinical data (18), the ORR was 45% in 76 patients with DLBCL with 24 CRs (32%), 57% in 14 patients with follicular lymphoma (five CR), 50% in four MCL patients (2 CR) (31). Here, we studied the pattern of activity of naratuximab emtansine across a large panel of cell lines derived from DLBCL and other lymphoma subtypes and characterized two resistance mechanisms to the ADC.

Enhancer RNAs (eRNAs) in Cancer: The Jacks of All Trades.

Enhancer RNAs (eRNAs) are non-coding RNAs (ncRNAs) transcribed in enhancer regions. They play an important role in transcriptional regulation, mainly during cellular differentiation. eRNAs are tightly tissue- and cell-type specific and are induced by specific stimuli, activating promoters of target genes in turn. eRNAs usually have a very short half-life but in some cases, once activated, they can be stably expressed and acquire additional functions. Due to their critical role, eRNAs are often dysregulated in cancer and growing number of interactions with chromatin modifiers, transcription factors, and splicing machinery have been described. Enhancer activation and eRNA transcription have particular relevance also in inflammatory response, placing the eRNAs at the interplay between cancer and immune cells. Here, we summarize all the possible molecular mechanisms recently reported in association with eRNAs activity.

Characterization of GECPAR, a noncoding RNA that regulates the transcriptional program of diffuse large B-cell lymphoma.

Enhancers are regulatory regions of DNA, which play a key role in cell-type specific differentiation and development. Most active enhancers are transcribed into enhancer RNA (eRNA) that can regulate transcription of target genes by means of in cis as well as in trans action. eRNA stabilize contacts between distal genomic regions and mediate the interaction of DNA with master transcription factors. Here, we characterized an enhancer eRNA, GECPAR (germinal center proliferative adapter RNA), which is specifically transcribed in normal and neoplastic germinal center B cells from the super-enhancer of POU2AF1, a key regulatory gene of the germinal center reaction. Using diffuse large B-cell lymphoma cell line models, we demonstrated the tumor suppressor activity of GECPAR, which is mediated via its transcriptional regulation of proliferation and differentiation genes, particularly MYC and the Wnt pathway.

Exon-Intron Differential Analysis Reveals the Role of Competing Endogenous RNAs in Post-Transcriptional Regulation of Translation.

Stressful conditions induce the cell to save energy and activate a rescue program modulated by mammalian target of rapamycin (mTOR). Along with transcriptional and translational regulation, the cell relies also on post-transcriptional modulation to quickly adapt the translation of essential proteins. MicroRNAs play an important role in the regulation of protein translation, and their availability is tightly regulated by RNA competing mechanisms often mediated by long noncoding RNAs (lncRNAs). In our paper, we simulated the response to growth adverse condition by bimiralisib, a dual PI3K/mTOR inhibitor, in diffuse large B cell lymphoma cell lines, and we studied post-transcriptional regulation by the differential analysis of exonic and intronic RNA expression. In particular, we observed the upregulation of a lncRNA, lncTNK2-2:1, which correlated with the stabilization of transcripts involved in the regulation of translation and DNA damage after bimiralisib treatment. We identified miR-21-3p as miRNA likely sponged by lncTNK2-2:1, with consequent stabilization of the mRNA of p53, which is a master regulator of cell growth in response to DNA damage.