T-bet suppresses proliferation of malignant B cells in chronic lymphocytic leukemia.

ABSTRACT: The T-box transcription factor T-bet is known as a master regulator of the T-cell response but its role in malignant B cells has not been sufficiently explored. Here, we conducted single-cell resolved multi-omics analyses of malignant B cells from patients with chronic lymphocytic leukemia (CLL) and studied a CLL mouse model with a genetic knockout of Tbx21. We found that T-bet acts as a tumor suppressor in malignant B cells by decreasing their proliferation rate. NF-κB activity, induced by inflammatory signals provided by the microenvironment, triggered T-bet expression, which affected promoter-proximal and distal chromatin coaccessibility and controlled a specific gene signature by mainly suppressing transcription. Gene set enrichment analysis identified a positive regulation of interferon signaling and negative control of proliferation by T-bet. In line, we showed that T-bet represses cell cycling and is associated with longer overall survival of patients with CLL. Our study uncovered a novel tumor suppressive role of T-bet in malignant B cells via its regulation of inflammatory processes and cell cycling, which has implications for the stratification and therapy of patients with CLL. Linking T-bet activity to inflammation explains the good prognostic role of genetic alterations in the inflammatory signaling pathways in CLL.

NRX-0492 degrades wild-type and C481 mutant BTK and demonstrates in vivo activity in CLL patient-derived xenografts.

Bruton tyrosine kinase (BTK) is essential for B-cell receptor (BCR) signaling, a driver of chronic lymphocytic leukemia (CLL). Covalent inhibitors bind C481 in the active site of BTK and have become a preferred CLL therapy. Disease progression on covalent BTK inhibitors is commonly associated with C481 mutations. Here, we investigated a targeted protein degrader, NRX-0492, that links a noncovalent BTK-binding domain to cereblon, an adaptor protein of the E3 ubiquitin ligase complex. NRX-0492 selectively catalyzes ubiquitylation and proteasomal degradation of BTK. In primary CLL cells, NRX-0492 induced rapid and sustained degradation of both wild-type and C481 mutant BTK at half maximal degradation concentration (DC50) of ≤0.2 nM and DC90 of ≤0.5 nM, respectively. Sustained degrader activity was maintained for at least 24 hours after washout and was equally observed in high-risk (deletion 17p) and standard-risk (deletion 13q only) CLL subtypes. In in vitro testing against treatment-naïve CLL samples, NRX-0492 was as effective as ibrutinib at inhibiting BCR-mediated signaling, transcriptional programs, and chemokine secretion. In patient-derived xenografts, orally administered NRX-0492 induced BTK degradation and inhibited activation and proliferation of CLL cells in blood and spleen and remained efficacious against primary C481S mutant CLL cells collected from a patient progressing on ibrutinib. Oral bioavailability, >90% degradation of BTK at subnanomolar concentrations, and sustained pharmacodynamic effects after drug clearance make this class of targeted protein degraders uniquely suitable for clinical translation, in particular as a strategy to overcome BTK inhibitor resistance. Clinical studies testing this approach have been initiated (NCT04830137, NCT05131022).

BTK inhibitors, irrespective of ITK inhibition, increase efficacy of a CD19/CD3-bispecific antibody in CLL.

Bruton tyrosine kinase inhibitors (BTKis) are a preferred treatment of patients with chronic lymphocytic leukemia (CLL). Indefinite therapy with BTKis, although effective, presents clinical challenges. Combination therapy can deepen responses, shorten treatment duration, and possibly prevent or overcome drug resistance. We previously reported on a CD19/CD3-bispecific antibody (bsAb) that recruits autologous T-cell cytotoxicity against CLL cells in vitro. Compared with observations with samples from treatment-naïve patients, T cells from patients being treated with ibrutinib expanded more rapidly and exerted superior cytotoxic activity in response to the bsAb. In addition to BTK, ibrutinib also inhibits interleukin-2 inducible T-cell kinase (ITK). In contrast, acalabrutinib, does not inhibit ITK. Whether ITK inhibition contributes to the observed immune effects is unknown. To better understand how BTKis modulate T-cell function and cytotoxic activity, we cultured peripheral blood mononuclear cells (PBMCs) from BTKi-naive and ibrutinib- or acalabrutinib-treated CLL patients with CD19/CD3 bsAb in vitro. T-cell expansion, activation, differentiation, and cytotoxicity were increased in PBMCs from patients on treatment with either BTKi compared with that observed for BKTi-naïve patients. BTKi therapy transcriptionally downregulated immunosuppressive effectors expressed by CLL cells, including cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and CD200. CTLA-4 blockade with ipilimumab in vitro increased the cytotoxic activity of the bsAb in BTKi-naïve but not BTKi-treated PBMCS. Taken together, BTKis enhance bsAb-induced cytotoxicity by relieving T cells of immunosuppressive restraints imposed by CLL cells. The benefit of combining bsAb immunotherapy with BTKis needs to be confirmed in clinical trials.

Effect of Bruton tyrosine kinase inhibitor on efficacy of adjuvanted recombinant hepatitis B and zoster vaccines.

Vaccinations are effective in preventing infections; however, it is unknown if patients with chronic lymphocytic leukemia (CLL) who are treatment naïve (TN) or receiving Bruton tyrosine kinase inhibitors (BTKi's) respond to novel adjuvanted vaccines. Understanding the effect of BTKi's on humoral immunity is timely because BTKi's are widely used and vaccination against coronavirus disease 2019 is urgently needed. In 2 open-label, single-arm clinical trials, we measured the effect of BTKi's on de novo immune response against recombinant hepatitis B vaccine (HepB-CpG) and recall response against recombinant zoster vaccine (RZV) in CLL patients who were TN or on BTKi. The primary end point was serologic response to HepB-CpG (anti-hepatitis B surface antibodies ≥10 mIU/mL) and RZV (≥fourfold increase in anti-glycoprotein E). The response rate to HepB-CpG was lower in patients on BTKi (3.8%; 95% confidence interval [CI], 0.7-18.9) than patients who were TN (28.1%; 95% CI, 15.6-45.4; P = .017). In contrast, the response rate to RZV did not differ significantly between the BTKi (41.5%; 95% CI, 27.8-56.6) and TN cohorts (59.1%; 95% CI, 38.7-76.7; P = .2). BTKi's were associated with a decreased de novo immune response following HepB-CpG, whereas recall immune response following RZV was not significantly affected by BTKi therapy. These trials were registered at www.clinicaltrials.gov as #NCT03685708 (Hep-CpG) and #NCT03702231 (RZV).

MARCKS affects cell motility and response to BTK inhibitors in CLL.

Bruton tyrosine kinase (BTK) inhibitors are highly active drugs for the treatment of chronic lymphocytic leukemia (CLL). To understand the response to BTK inhibitors on a molecular level, we performed (phospho)proteomic analyses under ibrutinib treatment. We identified 3466 proteins and 9184 phosphopeptides (representing 2854 proteins) in CLL cells exhibiting a physiological ratio of phosphorylated serines (pS), threonines (pT), and tyrosines (pY) (pS:pT:pY). Expression of 83 proteins differed between unmutated immunoglobulin heavy-chain variable region (IGHV) CLL (UM-CLL) and mutated IGHV CLL (M-CLL). Strikingly, UM-CLL cells showed higher basal phosphorylation levels than M-CLL samples. Effects of ibrutinib on protein phosphorylation levels were stronger in UM-CLL, especially on phosphorylated tyrosines. The differentially regulated phosphopeptides and proteins clustered in pathways regulating cell migration, motility, cytoskeleton composition, and survival. One protein, myristoylated alanine-rich C-kinase substrate (MARCKS), showed striking differences in expression and phosphorylation level in UM-CLL vs M-CLL. MARCKS sequesters phosphatidylinositol-4,5-bisphosphate, thereby affecting central signaling pathways and clustering of the B-cell receptor (BCR). Genetically induced loss of MARCKS significantly increased AKT signaling and migratory capacity. CD40L stimulation increased expression of MARCKS. BCR stimulation induced phosphorylation of MARCKS, which was reduced by BTK inhibitors. In line with our in vitro findings, low MARCKS expression is associated with significantly higher treatment-induced leukocytosis and more pronounced decrease of nodal disease in patients with CLL treated with acalabrutinib.

Clinical and biological implications of target occupancy in CLL treated with the BTK inhibitor acalabrutinib.

Inhibition of the B-cell receptor pathway, and specifically of Bruton tyrosine kinase (BTK), is a leading therapeutic strategy in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Target occupancy is a measure of covalent binding to BTK and has been applied as a pharmacodynamic parameter in clinical studies of BTK inhibitors. However, the kinetics of de novo BTK synthesis, which determines occupancy, and the relationship between occupancy, pathway inhibition and clinical outcomes remain undefined. This randomized phase 2 study investigated the safety, efficacy, and pharmacodynamics of a selective BTK inhibitor acalabrutinib at 100 mg twice daily (BID) or 200 mg once daily (QD) in 48 patients with relapsed/refractory or high-risk treatment-naïve CLL. Acalabrutinib was well tolerated and yielded an overall response rate (ORR) of partial response or better of 95.8% (95% confidence interval [CI], 78.9-99.9) and an estimated progression-free survival (PFS) rate at 24 months of 91.5% (95% CI, 70.0-97.8) with BID dosing and an ORR of 79.2% (95% CI, 57.9-92.9) and an estimated PFS rate at 24 months of 87.2% (95% CI, 57.2-96.7) with QD dosing. BTK resynthesis was faster in patients with CLL than in healthy volunteers. BID dosing maintained higher BTK occupancy and achieved more potent pathway inhibition compared with QD dosing. Small increments in occupancy attained by BID dosing relative to QD dosing compounded over time to augment downstream biological effects. The impact of BTK occupancy on long-term clinical outcomes remains to be determined. This trial was registered at www.clinicaltrials.gov as #NCT02337829.

Cardiovascular adverse events in patients with chronic lymphocytic leukemia receiving acalabrutinib monotherapy: pooled analysis of 762 patients.

Cardiovascular (CV) toxicities of the Bruton tyrosine kinase (BTK) inhibitor ibrutinib may limit use of this effective therapy in patients with chronic lymphocytic leukemia (CLL). Acalabrutinib is a second-generation BTK inhibitor with greater BTK selectivity. This analysis characterizes pooled CV adverse events (AE) data in patients with CLL who received acalabrutinib monotherapy in clinical trials (clinicaltrials gov. Identifier: NCT02029443, NCT02475681, NCT02970318 and NCT02337829). Acalabrutinib was given orally at total daily doses of 100-400 mg, later switched to 100 mg twice daily, and continued until disease progression or toxicity. Data from 762 patients (median age: 67 years [range, 32-89]; median follow-up: 25.9 months [range, 0-58.5]) were analyzed. Cardiac AE of any grade were reported in 129 patients (17%; grade ≥3, n=37 [5%]) and led to treatment discontinuation in seven patients (1%). The most common any-grade cardiac AE were atrial fibrillation/flutter (5%), palpitations (3%), and tachycardia (2%). Overall, 91% of patients with cardiac AE had CV risk factors before acalabrutinib treatment. Among 38 patients with atrial fibrillation/flutter events, seven (18%) had prior history of arrhythmia or atrial fibrillation/flutter. Hypertension AE were reported in 67 patients (9%), 43 (64%) of whom had a preexisting history of hypertension; no patients discontinued treatment due to hypertension. No sudden cardiac deaths were reported. Overall, these data demonstrate a low incidence of new-onset cardiac AE with acalabrutinib in patients with CLL. Findings from the head-to-head, randomized trial of ibrutinib and acalabrutinib in patients with highrisk CLL (clinicaltrials gov. Identifier: NCT02477696) prospectively assess differences in CV toxicity between the two agents.

A CD19/CD3 bispecific antibody for effective immunotherapy of chronic lymphocytic leukemia in the ibrutinib era.

The Bruton tyrosine kinase inhibitor ibrutinib induces high rates of clinical response in chronic lymphocytic leukemia (CLL). However, there remains a need for adjunct treatments to deepen response and to overcome drug resistance. Blinatumomab, a CD19/CD3 bispecific antibody (bsAb) designed in the BiTE (bispecific T-cell engager) format, is approved by the US Food and Drug Administration for the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia. Because of its short half-life of 2.1 hours, blinatumomab requires continuous intravenous dosing for efficacy. We developed a novel CD19/CD3 bsAb in the single-chain Fv-Fc format (CD19/CD3-scFv-Fc) with a half-life of ∼5 days. In in vitro experiments, both CD19/CD3-scFv-Fc and blinatumomab induced >90% killing of CLL cells from treatment-naïve patients. Antileukemic activity was associated with increased autologous CD8 and CD4 T-cell proliferation, activation, and granzyme B expression. In the NOD/SCID/IL2Rγnull patient-derived xenograft mouse model, once-weekly treatment with CD19/CD3-scFv-Fc eliminated >98% of treatment-naïve CLL cells in blood and spleen. By contrast, blinatumomab failed to induce a response, even when administered daily. We next explored the activity of CD19/CD3-scFv-Fc in the context of ibrutinib treatment and ibrutinib resistance. CD19/CD3-scFv-Fc induced more rapid killing of CLL cells from ibrutinib-treated patients than those from treatment-naïve patients. CD19/CD3-scFv-Fc also demonstrated potent activity against CLL cells from patients with acquired ibrutinib-resistance harboring BTK and/or PLCG2 mutations in vitro and in vivo using patient-derived xenograft models. Taken together, these data support investigation of CD19/CD3 bsAb's and other T cell-recruiting bsAb's as immunotherapies for CLL, especially in combination with ibrutinib or as rescue therapy in ibrutinib-resistant disease.

Depth and durability of response to ibrutinib in CLL: 5-year follow-up of a phase 2 study.

The safety and efficacy of ibrutinib (420 mg) in chronic lymphocytic leukemia (CLL) were evaluated in a phase 2 study; 51 patients had TP53 aberration (TP53 cohort) and 35 were enrolled because of age 65 years or older (elderly cohort). Both cohorts included patients with treatment-naive (TN) and relapsed/refractory (RR) CLL. With the median follow-up of 4.8 years, 49 (57.0%) of 86 patients remain on study. Treatment was discontinued for progressive disease in 20 (23.3%) patients and for adverse events in 5 (5.8%). Atrial fibrillation occurred in 18 (20.9%) patients for a rate of 6.4 per 100 patient-years. No serious bleeding occurred. The overall response rate at 6 months, the primary study endpoint, was 95.8% for the TP53 cohort (95% confidence interval, 85.7%-99.5%) and 93.9% for the elderly cohort (95% confidence interval, 79.8%-99.3%). Depth of response improved with time: at best response, 14 (29.2%) of 48 patients in the TP53 cohort and 9 (27.3%) of 33 in the elderly cohort achieved a complete response. Median minimal residual disease (MRD) in peripheral blood was 3.8 × 10-2 at 4 years, with MRD-negative (<10-4) remissions in 5 (10.2%) patients. In the TP53 cohort, the estimated 5-year progression-free survival (PFS) was 74.4% in TN-CLL compared with 19.4% in RR-CLL (P = .0002), and overall survival (OS) was 85.3% vs 53.7%, respectively (P = .023). In the elderly cohort, the estimated 5-year PFS and OS in RR-CLL were 64.8% and 71.6%, respectively, and no event occurred in TN-CLL. Long-term administration of ibrutinib was well tolerated and provided durable disease control for most patients. This trial was registered at www.clinicaltrials.gov as #NCT01500733.

Activation of Th1 Immunity within the Tumor Microenvironment Is Associated with Clinical Response to Lenalidomide in Chronic Lymphocytic Leukemia.

Immune stimulation contributes to lenalidomide's antitumor activity. Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of mature, autoreactive B cells in secondary lymphoid tissues, blood, and bone marrow and progressive immune dysfunction. Previous studies in CLL indicated that lenalidomide can repair defective T cell function in vitro. Whether T cell activation is required for clinical response to lenalidomide remains unclear. In this study, we report changes in the immune microenvironment in patients with CLL treated with single-agent lenalidomide and associate the immunologic effects of lenalidomide with antitumor response. Within days of starting lenalidomide, T cells increased in the tumor microenvironment and showed Th1-type polarization. Gene expression profiling of pretreatment and on-treatment lymph node biopsy specimens revealed upregulation of IFN-γ and many of its target genes in response to lenalidomide. The IFN-γ-mediated Th1 response was limited to patients achieving a clinical response defined by a reduction in lymphadenopathy. Deep sequencing of TCR genes revealed decreasing diversity of the T cell repertoire and an expansion of select clonotypes in responders. To validate our observations, we stimulated T cells and CLL cells with lenalidomide in culture and detected lenalidomide-dependent increases in T cell proliferation. Taken together, our data demonstrate that lenalidomide induced Th1 immunity in the lymph node that is associated with clinical response.

Harnessing the Effects of BTKi on T Cells for Effective Immunotherapy against CLL.

B-cell receptor (BCR) signaling and tumor-microenvironment crosstalk both drive chronic lymphocytic leukemia (CLL) pathogenesis. Within the microenvironment, tumor cells shape the T-cell compartment, which in turn supports tumor growth and survival. Targeting BCR signaling using Bruton tyrosine kinase inhibitors (BTKi) has become a highly successful treatment modality for CLL. Ibrutinib, the first-in-class BTKi, also inhibits Tec family kinases such as interleukin-2-inducible kinase (ITK), a proximal member of the T-cell receptor signaling cascade. It is increasingly recognized that ibrutinib modulates the T-cell compartment of patients with CLL. Understanding these T-cell changes is important for immunotherapy-based approaches aiming to increase the depth of response and to prevent or treat the emergence of resistant disease. Ibrutinib has been shown to improve T-cell function in CLL, resulting in the expansion of memory T cells, Th1 polarization, reduced expression of inhibitory receptors and improved immune synapse formation between T cells and CLL cells. Investigating the modulation of BTKi on the T-cell antitumoral function, and having a more complete understanding of changes in T cell behavior and function during treatment with BTKi therapy will inform the design of immunotherapy-based combination approaches and increase the efficacy of CLL therapy.

Partial reconstitution of humoral immunity and fewer infections in patients with chronic lymphocytic leukemia treated with ibrutinib.

Chronic lymphocytic leukemia (CLL) is characterized by immune dysregulation, often including hypogammaglobulinemia, which contributes to a high rate of infections and morbidity. Ibrutinib, a covalent inhibitor of Bruton tyrosine kinase (BTK), inhibits B-cell receptor signaling and is an effective, US Food and Drug Administration (FDA)-approved treatment of CLL. Inactivating germline mutations in BTK cause a severe B-cell defect and agammaglobulinemia. Therefore, we assessed the impact of ibrutinib on immunoglobulin levels, normal B cells, and infection rate in patients with CLL treated with single-agent ibrutinib on a phase 2 investigator-initiated trial. Consistent with previous reports, immunoglobulin G (IgG) levels remained stable during the first 6 months on treatment, but decreased thereafter. In contrast, there were a transient increase in IgM and a sustained increase in IgA (median increase 45% at 12 months, P < .0001). To distinguish the effects on clonal B cells from normal B cells, we measured serum free light chains (FLCs). In κ-clonal CLL cases, clonal (κ) FLCs were elevated at baseline and normalized by 6 months. Nonclonal (λ) FLCs, which were often depressed at baseline, increased, suggesting the recovery of normal B cells. Consistently, we observed normal B-cell precursors in the bone marrow and an increase in normal B-cell numbers in the peripheral blood. Patients with superior immune reconstitution, as defined by an increase in serum IgA of ≥50% from baseline to 12 months, had a significantly lower rate of infections (P = .03). These data indicate that ibrutinib allows for a clinically meaningful recovery of humoral immune function in patients with CLL. This trial was registered at www.clinicaltrials.gov as #NCT015007330.

Lymphocyte activation gene 3: a novel therapeutic target in chronic lymphocytic leukemia.

A novel therapeutic approach in cancer, attempting to stimulate host anti-tumor immunity, involves blocking of immune checkpoints. Lymphocyte activation gene 3 (LAG3) is an immune checkpoint receptor expressed on activated/exhausted T cells. When engaged by the major histocompatibility complex (MHC) class II molecules, LAG3 negatively regulates T-cell function, thereby contributing to tumor escape. Intriguingly, a soluble LAG3 variant activates both immune and malignant MHC class II-presenting cells. In the study herein, we examined the role of LAG3 in the pathogenesis of chronic lymphocytic leukemia, an MHC class II-presenting malignancy, and show that chronic lymphocytic leukemia cells express and secrete LAG3. High levels of surface and soluble LAG3 were associated with the unmutated immunoglobulin variable heavy chain leukemic subtype and a shorter median time from diagnosis to first treatment. Utilizing a mechanism mediated through MHC class II engagement, recombinant soluble LAG3-Ig fusion protein, LAG3-Fc, activated chronic lymphocytic leukemia cells, induced anti-apoptotic pathways and protected the cells from spontaneous apoptosis, effects mediated by SYK, BTK and MAPK signaling. Moreover, LAG3 blocking antibody enhanced in vitro T-cell activation. Our data suggest that soluble LAG3 promotes leukemic cell activation and anti-apoptotic effects through its engagement with MHC class II. Furthermore, MHC class II-presenting chronic lymphocytic leukemia cells may affect LAG3-presenting T cells and impose immune exhaustion on their microenvironment; hence, blocking LAG3-MHC class II interactions is a potential therapeutic target in chronic lymphocytic leukemia.

Incidence and risk factors of bleeding-related adverse events in patients with chronic lymphocytic leukemia treated with ibrutinib.

Ibrutinib is associated with bleeding-related adverse events of grade ≤ 2 in severity, and infrequently with grade ≥ 3 events. To investigate the mechanisms of bleeding and identify patients at risk, we prospectively assessed platelet function and coagulation factors in our investigator-initiated trial of single-agent ibrutinib for chronic lymphocytic leukemia. At a median follow-up of 24 months we recorded grade ≤ 2 bleeding-related adverse events in 55% of 85 patients. No grade ≥ 3 events occurred. Median time to event was 49 days. The cumulative incidence of an event plateaued by 6 months, suggesting that the risk of bleeding decreases with continued therapy. At baseline, von Willebrand factor and factor VIII levels were often high and normalized on treatment. Platelet function measured via the platelet function analyzer (PFA-100™) was impaired in 22 patients at baseline and in an additional 19 patients on ibrutinib (often transiently). Collagen and adenosine diphosphate induced platelet aggregation was tested using whole blood aggregometry. Compared to normal controls, response to both agonists was decreased in all patients with chronic lymphocytic leukemia, whether on ibrutinib or not. Compared to untreated chronic lymphocytic leukemia patients, response to collagen showed a mild further decrement on ibrutinib, while response to adenosine diphosphate improved. All parameters associated with a significantly increased risk of bleeding-related events were present at baseline, including prolonged epinephrine closure time (HR 2.74, P=0.012), lower levels of von Willebrand factor activity (HR 2.73, P=0.009) and factor VIII (HR 3.73, P=0.0004). In conclusion, both disease and treatment-related factors influence the risk of bleeding. Patients at greater risk for bleeding of grade ≤ 2 can be identified by clinical laboratory tests and counseled to avoid aspirin, non-steroidal anti-inflammatory drugs and fish oils. ClinicalTrials.gov identifier NCT01500733.

Prognosis and therapy of chronic lymphocytic leukemia and small lymphocytic lymphoma.

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course that has guided treatment principles in as much as anti-leukemic therapy is reserved for patients with active disease. This heterogeneity is somewhat dissected by prognostic markers, many of which represent pathogenic mechanisms. Recently, the introduction of highly active targeted agents and maturing data on predictive markers may lead to more individualized therapeutic approaches. In this chapter, we review key prognostic markers, current and emerging therapy, and will attempt to outline a future "where the two may connect".

Vaccinations in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by immune dysfunction resulting in heightened susceptibility to infections and elevated rates of morbidity and mortality. A key strategy to mitigate infection-related complications has been immunization against common pathogens. However, the immunocompromised status of CLL patients poses challenges in eliciting an adequate humoral and cellular immune response to vaccination. Most CLL-directed therapy disproportionately impairs humoral immunity. Vaccine responsiveness also depends on the phase and type of immune response triggered by immunization. In this review, we discuss the immune dysfunction, vaccine responsiveness, and considerations for optimizing vaccine response in patients with CLL.