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. BCR signaling has emerged as the pivotal pathway in the pathogenesis of both CLL and MCL. A major contribution from my group has been the first demonstration of active 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 patients. Since mouse models have implicated activation-induced cytidine deaminase in mutagenesis, we compared AICDA expression between cases with and without clonal evolution, but did not find a difference. In contrast, 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. The first-in-calls BTKi developed is ibrutinib. Between 2012 and 2014, our phase 2 clinical trial with ibrutinib enrolled 84 patients. Over twenty publications from my group report clinical and translational data from this particular study. We included 34 patients with TP53 alterations that were treated with single-agent ibrutinib. Historically, this groups of high-risk patients had a median survival of less than 3 years with intensive, multiagent chemoimmunotherapy. At a median follow-up of 6.5 years, 17 (50%) patients remained on study including six with complete responses. The most common reason for treatment discontinuation was disease progression (35%), followed by withdrawal of consents and deaths unrelated to CLL (6% each). Estimated 6-year progression-free (PFS) and overall survival was 61% and 79%, respectively. BTK and/or PLCG2 mutations were detected in 83% of progressors. We confirmed the long-term efficacy of ibrutinib in a pooled analysis of 89 patients with TP53 alterations. To improve tolerability and minimize off-target effects of ibrutinib, ACP-196 (acalabrutinib), a more BTK selective inhibitor was developed. Taking advantage of our PDX model, we contributed pre-clinical data supporting the translation of acalabrutinib into clinical trials. We investigated the safety, efficacy, and pharmacodynamics of acalabrutinib at 100 mg twice daily (BID) or 200 mg once daily (QD) in 48 patients with relapsed/refractory or high-risk treatment nave CLL. 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. Target occupancy, a measure of covalent binding of the drug to BTK, serves as a key pharmacodynamic parameter. The kinetics of de novo BTK synthesis, which determines durability of target occupancy, and the relationship between occupancy, pathway inhibition and clinical outcomes remained undefined. Facilitated by a pulse-chase design of the first week of the study, we could estimate BTK resynthesis rate at 15% per day. BID dosing maintained higher BTK occupancy and achieved more potent pathway inhibition compared to QD dosing. Small increments in occupancy attained by BID dosing relative to QD dosing compounded over time to augment downstream biological effects. In conclusion, acalabrutinib was an effective and safe treatment for relapsed or high-risk treatment nave CLL patients. Twice daily dosing increased BTK occupancy and the consequent downregulation of oncogenic pathways. Measurements of target occupancy support the twice daily dosing regimen approved by the FDA. Randomized trials established the superiority of ibrutinib-based therapy over chemoimmunotherapy in patients with CLL. PFS is quite variable based on pre-treatment factors. To develop a risk stratification tool that can assist in the selection of therapeutic strategies, we analyzed 804 CLL patients treated with ibrutinib across six clinical trials and correlated the clinical model with genetic biomarkers of ibrutinib resistance. Multivariate analysis and machine learning algorithms identified four factors for a prognostic model that was validated in internal and external cohorts. Factors independently associated with inferior PFS and OS were: TP53 aberration, prior treatment, -2 microglobulin 5mg/L, and lactate dehydrogenase >250U/L. Each of these four factors contributed one point to a prognostic model that stratified patients into three risk groups: 3-4 points, high-risk; 2 points, intermediate-risk; 0-1 point, low-risk. The 3-year PFS for all 804 patients combined was 47%, 74% and 87% for the high-, the intermediate, and the low-risk group, respectively (P<0.0001). The 3-year OS was 63%, 83% and 93%, respectively (P<0.0001). In conclusion, the 4-factor model defines high-risk disease among CLL patients treated with ibrutinib. Following up on our studies with ibrutinib in patients with TP53 aberration we investigated the prognostic value of carrying isolated (single-hit) or multiple (multi-hit) TP53 aberrations. In our cohort, we identified TP53 mutations in 43 patients (84%) and del(17p) in 47 (92%); 9 and 42 patients carried single-hit and multi-hit TP53, respectively. PFS was significantly shorter in patients with multi-hit TP53 compared with those with single-hit TP53. Thus, single-hit TP53 defines a distinct subgroup of patients with an excellent long-term response to single-agent ibrutinib To identify mechanisms of BTKi resistance in CLL, in addition to BTK and PLCG2 mutations, we collaborated with external groups. In vitro modeling of resistance to ibrutinib in the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma, which relies on chronic active BCR signaling for survival. The primary mode of resistance was epigenetic, driven in part by the transcription factor TCF4. A phenotypic shift altered BCR signaling such that the GTPase RAC2 substituted for BTK in activating downstream NF-B. We confirmed a role for RAC2 also in chronic lymphocytic leukemia cells from patients with persistent or progressive disease on BTKi. Clinically available drugs that can treat epigenetic ibrutinib resistance suggest possible therapeutic strategies.