Tumor microenvironment in CLL and MCL: pathogenesis, targets, and therapy

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are tumors of mature B cells that are closely related biologically and currently incurable. A major contribution from my group has been the first demonstration of active B-cell receptor (BCR) signaling in CLL patients in vivo. Furthermore, we showed that BCR signaling, and the consequent activation of the NF-kB pathway occurs primarily in the lymph node (LN) microenvironment rather than in the peripheral blood (PB) or bone marrow. Similarly, we found direct in vivo evidence for activation of the BCR and canonical NF-kB pathways in MCL that, in the absence of activating mutations, depends upon the LN microenvironment. These findings provide a mechanistic explanation for the surprising efficacy of Bruton Tyrosine Kinase inhibitors (BTKi) in treating MCL. To further characterize the intersection between microenvironment and molecular events in CLL pathogenesis, we integrated bulk transcriptome profiling of paired PB and LN samples from 34 patients. Oncogenic processes were upregulated in LN compared to PB. Single-cell RNA sequencing distinguished 3 major cell states: quiescent, activated, and proliferating. The activated subpopulation comprised only 2.2% to 4.3% of the total tumor bulk in LN samples. RNA velocity analysis revealed that CLL cell fate in LN is unidirectional, starts in the proliferating state, transitions to the activated state, and ends in the quiescent state. A 10-gene signature derived from activated tumor cells was associated with inferior treatment-free survival and positively correlated with the proportion of activated CD4+ memory T cells and M2 macrophages in LN. Whole exome sequencing of paired PB and LN samples showed subclonal expansion in LN in approximately half of the patients. The presence of a T-cell inflamed microenvironment in LN was associated with clonal stability. In summary, a distinct minor tumor subpopulation underlies CLL pathogenesis and drives clinical outcome. Clonal trajectories are shaped by the LN milieu where T-cell immunity may contribute to suppress clonal outgrowth. Small molecule inhibitors of BCR signaling have transformed clinical approaches. Between 2012 and 2014, our phase 2 clinical trial with ibrutinib enrolled 84 patients. We included 34 patients with TP53 alterations that were treated with single-agent ibrutinib. At a median follow-up of 6.5 years, 17 (50%) patients remained on study including six with complete responses. Estimated 6-year progression-free (PFS) and overall survival was 61% and 79%, respectively. We contributed to a multicenter analysis of first-line ibrutinib for 89 patients with TP53 aberrations that confirmed our single center experience. The overall response rate was 93%, including complete response in 39% of patients. With a median follow-up of 498 months, median PFS was not reached. PFS and OS at 4 years were estimated at 79% and 88%, respectively. In our phase 2 study, we identified TP53 mutations in 43 (84%) of 51 patients and del(17p) in 47 (92%); 9 and 42 patients carried single-hit and multi-hit TP53 alterations, respectively. The multi-hit TP53 subgroup was enriched with younger patients who had U-CLL and a history of prior treatments. OS and PFS were significantly shorter in patients with multi-hit TP53 compared with those with single-hit TP53 alterations. Results were validated in an independent population-based cohort of 112 patients with CLL treated with ibrutinib. To improve tolerability and minimize off-target effects of ibrutinib, ACP-196 (acalabrutinib), a more BTK selective inhibitor was developed. Acalabrutinib was well tolerated with over 90% of patients responding and an estimated PFS rate at 24 months of 79.2% and 91.5% with QD and BID dosing, respectively. To determine the role of CD49d for response to BTKi in patients with CLL we measured CD49d expression on CLL cells and its activation state and correlated these data with treatment response of patients enrolled on our ibrutinib and acalabrutinib studies. In the combined cohorts of 121 BTKi treated patients, 48 (39.7%) progressed on treatment with BTK and/or PLCG2 mutations detected in 87% of CLL progressions. Consistent with a recent report, homogeneous and bimodal CD49d positive cases (the latter having concurrent CD49d+ and CD49d- CLL subpopulations, irrespective of the traditional 30% cutoff), had a shorter time to progression of 6.6 years, while 90% of cases homogenously CD49d negative were estimated progression-free at 8 years (P = .0004). In patients treated with acalabrutinib, treatment induced lymphocytosis was comparable for both subgroups but resolved more rapidly for CD49d+ cases. Acalabrutinib inhibited constitutive VLA-4 activation but was insufficient to block BCR and CXCR4 mediated inside-out activation. Transcriptomes of CD49d+ and CD49d- cases were compared using RNA sequencing at baseline and at 1 and 6 months on treatment. Gene set enrichment analysis revealed increased constitutive NF-B and JAK-STAT signaling, enhanced survival, adhesion and migratory capacity in CD49d+ over CD49- CLL that was maintained during therapy. In conclusion, CD49d/VLA-4 emerged as a microenvironmental factor that contributes to BTKi resistance in CLL. The prognostic value of CD49d is improved by considering bimodal CD49d expression. Given the strong impact of CD49d on outcomes with BTKi therapy, we explored whether CD49d could independently add to the prognostic information of our previously described 4-factor model. The 4 factors independently associated with inferior PFS and OS were presence of TP53 aberration, prior treatment, -2 microglobulin 5mg/L, and lactate dehydrogenase >250U/L. Each of these 4 factors contributed one point to a prognostic model that stratified patients into high (3 or 4 points), intermediate (2 points), and low (0-1 point) risk. The 3-year PFS for all 804 patients combined was 47%, 74% and 87% for the high, intermediate, and low risk disease, respectively (P<.0001). In multivariate analysis, CD49d expression remained significantly associated with progression (adjusted HR= 4.99, P=.007). In particular, among 90 patients classified as low and intermediate risk by the CLL-4 model, CD49d expression separated groups with starkly different risks of progression (P =.003). While these data suggest that adding CD49d to a 5-factor model would increase the prognostic significance, this should be tested systematically in larger cohorts and include external validation. Unfortunately, CD49d testing has not been common in routine testing, but, arguably, it should be added prospectively given its biologic role and value as a prognostic marker. To overcome resistance to BTKi, we investigated BTK degradation as another promising approach to target mutant BTK. We provided preclinical proof of concept using NRX-0492, a targeted protein degrader, in preclinical models. NRX-0492 consists of noncovalent BTK-binding hook linked to a harness that recruits cereblon, an adaptor protein of the E3 ubiquitin ligase complex (Fig. 7). NRX-0492 catalyzed ubiquitylation and proteasomal degradation of both wild-type and C481S mutant BTK at low nanomolar concentrations in less than 24 hours. We also tested NRX-0492 against ibrutinib resistant CLL using samples from a patient who had been on ibrutinib for over 7 years before progressing with C481S mutations in 88% of the tumor cells. Drug-induced BTK degradation and inhibition of proliferation was demonstrated. We opened a clinical trial with NX-2127, a related compound that has been further optimized for clinical use. Initial observations in patients treated with NX-2127 are very promising.