Mutations In Chronic Lymphocytic Leukemia Research Articles

Resisting the Resistance: Navigating BTK Mutations in Chronic Lymphocytic Leukemia (CLL).

Bruton's tyrosine kinase (BTK) plays a key role in the B-cell receptor (BCR) signaling pathway and confers anti-apoptotic and proliferative properties to malignant B-cells in chronic lymphocytic leukemia (CLL). Small molecule BTK inhibitors were designed to bind BTK's active site and block downstream signaling. These drugs have now been used in the treatment of thousands of patients with CLL, the most common form of leukemia in the western hemisphere. However, adverse effects of early generations of BTK inhibitors and resistance to treatment have led to the development of newer, more selective and non-covalent BTK inhibitors. As the use of these newer generation BTK inhibitors has increased, novel BTK resistance mutations have come to light. This review aims to discuss previously known and novel BTK mutations, their mechanisms of resistance, and their relationship with patient treatment. Also discussed here are future studies that are needed to investigate the underlying cause allowing these mutations to occur and how they incite resistance. New treatments on the horizon that attempt to maneuver around these resistance mutations can be met with new resistance mutations, creating an unmet need for patients with CLL. Novel therapies and combinations that address all forms of resistance are discussed.

A First-in-Human Phase 1 Trial of NX-2127, a First-in-Class Bruton's Tyrosine Kinase (BTK) Dual-Targeted Protein Degrader with Immunomodulatory Activity, in Patients with Relapsed/Refractory B Cell Malignancies

Introduction: Although BTK inhibitors (BTKi) are effective therapeutics in the treatment of B cell malignancies, emerging BTK resistance mutations in chronic lymphocytic leukemia (CLL), as well as potential growth-promoting kinase-independent scaffolding function of BTK, present a need for improved or new approaches. Additionally, preclinical and clinical data in non-Hodgkin's lymphoma (NHL) suggest that drugs modulating cereblon may synergize with BTKi to provide a therapeutic effect. NX-2127 is an oral, first-in-class, dual-function small molecule degrader that combines BTK degradation with the immunomodulatory activity of an Ikaros and Aiolos degrader. Preliminary safety of NX-2127 in patients across B cell malignancies and efficacy in patients with CLL have been presented previously [Mato et al. 2022; Danilov et al. 2023]. Here we report further safety and efficacy follow-up in patients with CLL and efficacy data in patients with NHL enrolled to date. Methods: NX-2127-001 (NCT04830137) is a first-in-human, multicenter, open-label, dose-escalation (Phase 1a) and cohort-expansion (Phase 1b) trial evaluating the safety and preliminary efficacy of NX-2127 in adults with relapsed/refractory B cell malignancies, including CLL, diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and Waldenstrom's macroglobulinemia (WM). NX-2127 is administered orally once daily in 28-day cycles. Results: As of 9 June 2023, 47 patients were enrolled and treated with NX-2127 at once-daily doses of 100 mg (n=28), 200 mg (n=10), and 300 mg (n=9). Patients were predominantly male (66%), with a median age of 74 (range 50-92) years. Twenty-nine patients were treated for CLL/small lymphocytic lymphoma, 5 DLBCL (2 GCB [Germinal-center B-cell-like], 3 non-GCB), 5 MCL, 3 MZL, 3 WM, and 2 FL. Patients enrolled were heavily pretreated with median prior lines of therapy of 4 (range 2-10) in NHL and 5 (range 2-11) in CLL. Prior treatments in patients with CLL comprised: BTKi 100% (including covalent [cBTKi] and non-covalent [ncBTKi] inhibitors); BCL2 inhibitor 76%, with a large proportion of CLL patients exhibiting BTKi resistance mutations at baseline. Prior treatments in patients with NHL included: BTKi 72% (cBTKi and ncBTKi); bispecific antibody 1/18; bispecific antibody and CAR-T 1/18. There were two reported dose-limiting toxicities: one previously reported cognitive disturbance in a patient with CLL treated at 300 mg; and neutropenia in a patient with MZL treated at 300 mg. The most common any grade treatment-emergent adverse events (TEAEs) were fatigue (48.9%), neutropenia (42.6%) and hypertension (36.2%, see Table). The most common grade ≥3 TEAEs were neutropenia (38.3%), hypertension (14.9%) and anemia (12.8%). Contusion was reported in 27.7% of patients (all below grade 3), atrial fibrillation in 12.8% of patients (6.4% grade ≥3). Most common reasons for treatment discontinuation were progressive disease (PD, 25.5%) and AE (21.3%). Median follow-up for the study was 9.5 (range 0.1-24.3) months. NX-2127 exhibited dose-dependent pharmacokinetics (PK) with a mean half-life of 2-4 days across cohorts. Rapid, robust and sustained BTK degradation was observed in all patients, regardless of their absolute BTK starting level, tumor type, or dose level of NX-2127 (Figure). In addition, degradation of the cereblon neo-substrate Ikaros was observed. Among the efficacy evaluable patients with CLL, there were 9 PRs/PR with rebound lymphocytosis; additionally, 11 patients had SD at the time of data cut-off and 4 had PD. Two patients with WM were treated and efficacy evaluable (1 SD, 1 PD). Among the efficacy evaluable patients with NHL, there were 2 CRs and 1 PR; additionally, 3 patients had SD, and 5 had PD. Two CRs are ongoing with 9.2 and 11.8 months of duration. Conclusion: This first-in-human study of NX-2127 is actively enrolling and dose-expansion cohorts in DLBCL and MCL have been initiated at the 300 mg daily dose. Findings include dose-dependent PK accompanied by degradation of BTK and Ikaros. Encouraging and persistent responses were observed in heavily pretreated patients with relapsed/refractory CLL and NHL with a manageable safety profile. These data support the treatment concept of combining immunomodulatory activity and BTK degradation in a single molecule and support further development of NX-2127 in B cell malignancies.

Initial Findings from a First-in-Human Phase 1a/b Trial of NX-5948, a Selective Bruton's Tyrosine Kinase (BTK) Degrader, in Patients with Relapsed/Refractory B Cell Malignancies

Introduction: Although BTK inhibitors (BTKi) are effective therapeutics in the treatment of B cell malignancies, emerging BTK resistance mutations in chronic lymphocytic leukemia (CLL), as well as potential growth-promoting kinase-independent scaffolding function of BTK, present a need for improved or new approaches. NX-5948 is a novel, orally administered small molecule that induces specific BTK protein degradation by the cereblon E3 ligase complex without degradation of other cereblon neo-substrates. Importantly, NX-5948 induces degradation of wild-type and mutant forms of BTK in B-cells [Noviski et al. 2023] at sub-nanomolar potencies and exhibits potent tumor growth inhibition in TMD8 xenograft models that contain either wild-type BTK or BTKi-resistant mutations [Robbins 2021]. Here we provide the first disclosure of preliminary safety and efficacy findings from a Phase 1a trial of NX-5948 in patients with relapsed/refractory B cell malignancies. Methods: NX-5948-301 is a Phase 1, first-in-human, dose-escalation and cohort-expansion trial evaluating the safety, tolerability, and clinical activity of NX-5948 in relapsed/refractory CLL and various subtypes of non-Hodgkin's lymphoma (NHL). Key eligibility criteria: ≥2 prior lines of therapy; measurable or other evaluable disease per indication-specific response criteria; Eastern Cooperative Oncology Group Performance Status (ECOG PS) 0-1. Phase 1a (dose escalation) evaluates safety and tolerability of NX-5948 via a standard 3+3 dose escalation in patients with relapsed/refractory B cell malignancies. Approximately 110 patients (30 in Phase 1a, 80 in Phase 1b) may be enrolled and treated until confirmed disease progression or unacceptable toxicity. Endpoints include dose-limiting toxicities (DLTs); treatment-emergent adverse events (TEAEs); deaths; changes in safety parameters; objective response rate per disease-specific response criteria. Phase 1b (dose expansion) will include up to four expansion cohorts. Results: As of June 9, 2023, 14 patients were enrolled in Phase 1a and received NX-5948 at 50 mg (n=7), 100 mg (n=4), or 200 mg (n=3) orally once daily. Median age was 65 (range 46-79) years; female/male ratio 28.6%/71.4%; white 92.9%; ECOG PS 0/1 21.4%/78.6%; primary diagnoses were CLL (n=4), diffuse large B cell lymphoma (DLBCL, n=4), mantle cell lymphoma (MCL, n=3), marginal zone lymphoma (MZL, n=2), and follicular lymphoma (FL, n=1). Median number of prior therapies was 4.5 (range 2-10), which included: for CLL - BTKi (n=4/4) and BCL2 inhibitor (n=3/4); for NHL - BTKi (n=5/10), bispecific antibody (n=3/10), and CAR-T (n=2/10). NX-5948 was well tolerated with no DLTs and no TEAEs resulting in drug discontinuation or dose reduction. In addition, there were no NX-5948-related grade ≥3 TEAEs or related serious adverse events. The most common TEAEs were purpura/contusion (57.1%, all below grade 3), nausea (35.7%), and thrombocytopenia (35.7%). No atrial fibrillation/flutter or hypertension was reported. Median duration of treatment was 2.8 (range 0.5-9.6) months with 9/14 patients remaining on treatment. Current data indicate that NX-5948 exhibits dose-dependent pharmacokinetics (PK) and a half-life of ~24 hours, supporting once daily dosing (Figure a). Rapid, robust and sustained BTK degradation was observed in all patients, regardless of absolute BTK starting level, tumor type, or NX-5948 dose (Figure b). Of three evaluable patients with CLL receiving the lowest dose of 50 mg, early signs of clinical activity were observed including one confirmed partial response (PR; at 8 and 16 weeks) and 2 patients with stable disease (SD; at 8 weeks). Further treatment responses will be reported at the time of presentation. Summary/conclusion: Current findings in this heavily pre-treated population of patients with CLL and NHL are encouraging and indicate that NX-5948 is safe and well tolerated and has clinical activity, supporting continuation of its development in CLL and NHL. NX-5948 also exhibits dose-proportional PK, resulting in rapid, robust and sustained BTK degradation. Additional data with higher dose levels and longer treatment duration will be presented at the meeting.

Higher Mutational Burden Is an Independent Predictor of Shorter Time to First Treatment in Untreated Chronic Lymphocytic Leukemia Patients

Background. Recent advancements in next-generation sequencing (NGS) technologies have revolutionized the ability to comprehensively profile chronic lymphocytic leukemia (CLL), unveiling various molecular lesions. Complex mutational landscapes and diverse combinatorial patterns of genetic alterations lead to heterogeneity of clinical course and outcomes. The primary objective of this study was to identify patterns of co-occurrence (CO) and mutual exclusivity (ME) among mutations in CLL and their impact on time to first treatment (TTFT). Methods. The cohort included 795 treatment-naïve (TN) patients (pts) with CLL diagnosed according to iwCLL guidelines. The median follow-up time for the cohort was 3.91 years (range 0 - 42.8). The median time from diagnosis to mutational analysis was 6.6 months (26.50 - 33.27, 95% CI). Mutational screening was performed by a clinical targeted NGS panel, covering all or hotspot exons of 95 genes - the Rapid Heme Panel implemented by BWH (Kluk MJ, et al. J Mol Diagn. 2016;18(4):507-515). Standard protocols were applied to analyze IGHV gene mutational status, chromosomal aberrations by FISH with probes for 11q, 13q, 17p, and trisomy 12, and stimulated karyotype, where complex karyotype (CK) was defined as ≥3 abnormalities. CO and ME patterns were analyzed in pts with a higher mutational burden, determined as ≥2 distinct mutated genes, using Fisher's exact test ( p<.05). Control for false discovery rate was performed using the Benjamini, Krieger and Yekutieli method ( q=20%). TTFT was the time from diagnosis to initial treatment. Results. We detected 1798 mutations in 719 (90.4%) pts. No mutations were detected in 9.6% (n=76) patients, while a single mutated gene was found in 216 (27.2%) pts, 2 mutated genes in 206 (25.9%) pts, and mutations in 3 and ≥4 genes were found in 153 (19.2%) and 144 (18.1%) pts, respectively. The most mutated (≥10%) genes in the cohort were ATM (18.1%), NOTCH1 (17.7%), TP53 (13.2%), SF3B1 (12.3%), TET2 (10.2%), and DNMT3A (10.1%). A higher mutational burden was associated with clinical Rai stages 1-4 [ p=.003]; elevated (>3.5 mg/L) levels of baseline ß-2 microglobulin (B2M) [ p=.036] and established high-risk genomic features: del(17p) [ p<.001], del(11q) [ p=.021], CK [ p=.009], unfavorable ZAP70 [ p=.027] and unmutated IGHV (UM- IGHV) [ p<.001] (incidence of UM- IGHV cases is 48.4% (n=359) in cohort). At the same time, cases with 0-1 mutation were more likely to have del(13q) [ p=.006]. Sex and age >65 years at diagnosis were not associated with mutational burden. Otherwise, lack of any mutations was associated with absence of del(17p) (0% vs 11.1% [ p=.002]), lower rates of B2M >3.5 mg/L (3.9% vs 21.7% [ p<.001]), less frequent UM- IGHV (25.4% vs 50.9% [p<.001]), lower ratio of CK (7.0% vs 17.5% [p=.024] and age >65 years at diagnosis (21.1% vs 35.1% [ p=.014]). After controlling for multiple comparisons, no genes showed significant mutational co-occurrence. UM- IGHV cases were enriched in mutated ATM [ p=.003], NOTCH1 [ p<.00001], SF3B1 [ p=.0012] and XPO1 [ p<.00001]. Mutated NOTCH1 [ p<.00001], as well as KRAS [ p=.0012], occurred along with tri12. In contrast, UM- IGHV did not co-occur with mutations in MYD88 [ p<.00001], as previously reported. Del(13q) did not co-occur with mutated BRAF [ p=.0006] or BCOR [ p=.006]. Mutated TP53 [ p=.008] and SF3B1 [ p=.0013] were absent in tri12 positive cases. Increased mutational burden in ≥2 distinct mutated genes predicted a shorter TTFT than 0 or 1 mutated gene (HR 2.16; 95%CI 1.57 - 2.97; p<.001). Factors including Rai stages 1-4 (HR 2.63; 95%CI 1.88 - 3.7; p<0.001), UM- IGHV (HR 2.4; 95%CI 1.74 - 3.3; p<.001), CK (HR 1.47; 95%CI 1.07 - 2.0; p=.018), mutational burden in ≥2 genes (HR 1.57; 95%CI 1.14 - 2.2; p=.006) and del(17p) /TP53mut (HR 1.79; 95%CI 1.15 - 2.8; p=.01) were independently associated with worsened TTFT ( Figure 1). Neither mutated TP53 without del(17p) (HR 1.47; 95%CI 0.92 - 2.3; p=.104), or wild-type TP53 with del(17p) (HR 1.03; 95%CI 0.51 - 2.1; p=.932) were significant predictors of TTFT. Conclusions: Higher mutational burden is associated with worsened TTFT in CLL pts independently of other genomic events. In this cohort we did not see co-occurrence of specific mutations.

Researching New Genetic Mutations in Chronic Lymphocytic Leukemia

Researching New Genetic Mutations in Chronic Lymphocytic Leukemia

The Broad Spectrum of TP53 Mutations in CLL: Evidence of Multiclonality and Novel Mutation Hotspots

TP53 aberrations are a major predictive factor of resistance to chemoimmunotherapy in chronic lymphocytic leukemia (CLL), and an assessment of them before each line of treatment is required for theranostic stratification. Acquisition of subclonal TP53 abnormalities underlies the evolution of CLL. To better characterize the distribution, combination, and impact of TP53 variants in CLL, 1,056 TP53 variants collected from 683 patients included in a multicenter collaborative study in France were analyzed and compared to UMD_CLL, a dataset built from published articles collectively providing 5,173 TP53 variants detected in 3,808 patients. Our analysis confirmed the presence of several CLL-specific hotspot mutations, including a two-base pair deletion in codon 209 and a missense variant at codon 234, the latter being associated with alkylating treatment. Our analysis also identified a novel CLL-specific variant in the splice acceptor signal of intron 6 leading to the use of a cryptic splice site, similarly utilized by TP53 to generate p53psi, a naturally truncated p53 isoform localized in the mitochondria. Examination of both UMD_CLL and several recently released large-scale genomic analyses of CLL patients confirmed that this splice variant is highly enriched in this disease when compared to other cancer types. Using a TP53-specific single-nucleotide polymorphism, we also confirmed that copy-neutral loss of heterozygosity is frequent in CLL. This event can lead to misinterpretation of TP53 status. Unlike other cancers, CLL displayed a high proportion of patients harboring multiple TP53 variants. Using both in silico analysis and single molecule smart sequencing, we demonstrated the coexistence of distinct subclones harboring mutations on distinct alleles. In summary, our study provides a detailed TP53 mutational architecture in CLL and gives insights into how treatments may shape the genetic landscape of CLL patients.

Different prognostic impact of recurrent gene mutations in chronic lymphocytic leukemia depending on IGHV gene somatic hypermutation status: a study by ERIC in HARMONY

Recent evidence suggests that the prognostic impact of gene mutations in patients with chronic lymphocytic leukemia (CLL) may differ depending on the immunoglobulin heavy variable (IGHV) gene somatic hypermutation (SHM) status. In this study, we assessed the impact of nine recurrently mutated genes (BIRC3, EGR2, MYD88, NFKBIE, NOTCH1, POT1, SF3B1, TP53, and XPO1) in pre-treatment samples from 4580 patients with CLL, using time-to-first-treatment (TTFT) as the primary end-point in relation to IGHV gene SHM status. Mutations were detected in 1588 (34.7%) patients at frequencies ranging from 2.3–9.8% with mutations in NOTCH1 being the most frequent. In both univariate and multivariate analyses, mutations in all genes except MYD88 were associated with a significantly shorter TTFT. In multivariate analysis of Binet stage A patients, performed separately for IGHV-mutated (M-CLL) and unmutated CLL (U-CLL), a different spectrum of gene alterations independently predicted short TTFT within the two subgroups. While SF3B1 and XPO1 mutations were independent prognostic variables in both U-CLL and M-CLL, TP53, BIRC3 and EGR2 aberrations were significant predictors only in U-CLL, and NOTCH1 and NFKBIE only in M-CLL. Our findings underscore the need for a compartmentalized approach to identify high-risk patients, particularly among M-CLL patients, with potential implications for stratified management.

Treatment Patterns and Outcomes of Patients with Chronic Lymphocytic Leukemia with TP53 Mutations

Background: Aberration of the TP53 gene in chronic lymphocytic leukemia (CLL) is a well-established marker of poor prognosis and a predictor of chemorefractory disease. Identification of a TP53 gene mutation is clinically useful in determining which patients may benefit from small-molecule inhibitors such as Bruton tyrosine kinase (BTKi) and BCL2 inhibitors (BCL2i). However, the treatment patterns in routine practice and outcomes of patients harboring TP53 mutations in CLL is not well reported. We present a descriptive analysis of CLL patients treated at Mayo Clinic with TP53 mutations including their demographics, treatment patterns, and outcomes. Methods: Patients with CLL who had a TP53 mutation detected by Sanger sequencing between June 2014 and April 2021 at Mayo Clinic Rochester were identified from a hematopathology database. Patient demographics, clinical and pathologic characteristics, treatment, and follow-up information were abstracted from the Mayo Clinic CLL database and by chart review. Date of TP53 mutation test was baseline. Discrete variables were compared using Chi-square or Fisher's exact tests; continuous variables were compared using Kruskal-Wallis test. Overall survival (OS) was calculated from date of TP53 mutation test until date of death from any cause or last known date alive. Rates of Richter transformation (RT) by prior treatment exposure were estimated by cumulative incidence accounting for competing risk of death. Results: A total of 145 patients were identified; 70% were male, and the median age at diagnosis was 59 years (range: 36-92). At diagnosis, 15% of patients had Rai stage III-IV, 77% had unmutated IGHV, 38% had del17p, 92% had high or very high CLL-IPI, and 62% had ≥3 cytogenetic abnormalities (Table 1). A TP53 gene mutation was identified prior to any CLL treatment in 63 patients (43%, Group 1) and after at least one prior line of treatment in 82 patients (57%, Group 2). The median time from CLL diagnosis to the first detected TP53 mutation was 3 months in Group 1 and 96 months in Group 2. There was significant heterogeneity in types of TP53 mutations with the majority of variants (n=102) being unique to an individual. At the time of TP53 mutation detection, Group 2 patients more frequently had advanced Rai stage and unmutated IGHV status (Table 1). Del17p by FISH was present prior to or at time of TP53 mutation identification (+/- 3 months) in 54% of patients in Group 1 and 52% in Group 2. Group 1 had a median follow-up of 26 months (from TP53 mutation detection date); 46/63 patients had initiated a first-line (1L) treatment (39 BTKi-based, 1 BCL2i-based, 2 BTKi+BCL2i-based, 4 chemoimmunotherapy [CIT]). In Group 2, the median number of prior lines of therapy was 2 (range 1-16). Prior treatment exposure included 38 (46%) CIT only, 41 (50%) BTKi-based ± CIT, and 3 (4%) BTKi-based + BCL2i-based ± CIT. Median follow-up was 21 months; 56/82 patients had initiated a new line of therapy (subsequent 1L). For 38 patients with prior CIT only exposure, 28 had subsequent 1L including 21 BTKi-based, 2 BCL2i-based, and 5 CIT. For 44 patients with prior BTKi exposure, 28 had subsequent 1L including 5 BTKi-based, 5 BCL2i-based, 9 BTKi+BCL2i-based, and 9 CIT. The median OS was not reached in Group 1 and was 40.6 months (95% CI 30.1-NE) in Group 2 (HR=0.21, 95% CI=0.10-0.45) (Figure 1). In Group 2, the median OS for patients with prior CIT only exposure was 55.5 (95% CI 30.1-NE) months and for patients with prior BTKi exposure was 33.3 (95% CI 14.7-NE) months (HR=0.59, 95% CI=0.31-1.12) (Figure 1). Three patients in Group 1 and 13 patients in Group 2 (3 prior CIT only, 10 prior BTKi) developed RT during the follow-up period; the cumulative incidence rates of RT at 2- and 5-year were 1.7% and 4.6%, respectively, in Group 1, and 10.8% and 21.0%, respectively, in Group 2. Conclusions: Regardless of prior treatment exposure, enrichment for high-risk features was present at time of TP53 mutation detection. Majority of patients were treated with novel agents after known TP53 mutations. OS after TP53 mutation was inferior in patients who had been previously treated, particularly those who had BTKi exposure already prior to TP53 mutation detection, compared to previously untreated patients. Future studies are needed to understand the characteristics of clonal evolution before and after treatment and the clinical impact of specific TP53 mutations. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Disrupting MGA-MYC Driven Metabolic Reprogramming in Richter's Syndrome Pre-Clinical Models Via Novel Therapeutic Approaches

Richter's syndrome (RS) is an aggressive transition of chronic lymphocytic leukemia (CLL) to lymphoma; however, molecular mechanisms underlying CLL-to-RS transformation are poorly understood. MYC network alterations are observed in 70% of RS cases, and MGA (Max-gene-associated), a functional MYC suppressor, undergoes loss-of-function mutations in ~7-20% of RS cases. MYC overexpression induces oxidative stress via reactive oxygen species (ROS) in several B-cell lymphomas. Using CRISPR-Cas9, we recently developed a B-cell restricted CLL to RS murine model by engineering loss-of-function Mga mutations in early progenitors (LSK) in the presence of commonly occurring CLL mutations 13q deletion (Mdr) and Sf3b1K700E. We found Myc and Nme1 (Nucleoside diphosphate kinase), both common targets of Mga, were up-regulated, altering the mitochondrial structure, and increasing oxidative phosphorylation (OXPHOS), leading to mitochondrial dysregulation. Based on these observations, we sought to understand the molecular basis of mitochondrial dysregulation and determine if targeting the Mga-Myc-Nme1 axis is beneficial for RS in vivo. Dysregulated mitochondria produce reactive oxygen species (ROS), byproducts of OXPHOS that are reported to trigger oncogenic signaling. To determine the molecular basis of mitochondrial dysregulation, we used our previously established Mga KO (knockout) driven CLL to RS murine model, which develops RS upon CLL cells transplantation into either immunocompetent or immunocompromised mice with high Ki67, MYC, and CD5 loss along with aberrant changes in mitochondria. Firstly, we measured cellular and mitochondrial ROS in splenic B cells derived from CLL, RS, and healthy control mice (n=3 each). CLL and RS B cells had higher cellular ROS and mitochondrial ROS than normal B cells. RS cells showed an elevated cellular ROS compared to CLL cells (p< 0.001). Accordingly, RS mitochondria exhibited high utilization of TCA cycle substrates such as succinate and malate (Seahorse substrate oxidation test). The substrates for complex II of the mitochondrial electron transport chain were also highly utilized. To unravel the molecular mechanism underlying MGA KO-induced metabolic changes, we generated MGA KO human B cell lines [MEC1, HG3, Nalm6E (harboring SF3B1K700E)] by CRISPR-Cas9. MGA KO increased cellular ROS in all cell lines and upregulated NME1, ETC (electron transport chain) complex II protein levels, suggesting that NME1, and ETC complex II may regulate ROS. Moreover, MGA KO induced pERK1/2 (ERK) and p4E-BP1 (mTOR) activation in an NME1-dependent fashion, which was reversed by antioxidant treatment (N-acetyl cysteine), highlighting MGA KO-induced ROS as a key driver of oncogenic activation. Given that ROS activates oncogenic signaling in MGA KO B cells, we exploited mitochondrial dysregulation as a potential therapeutic approach to treating RS. Firstly, we tested the inhibitor of complex II (TTFA: Thenoyltrifluoroacetone) and CDK9 (AZ5576) in MGA KO Nalm6E cells. TTFA and CDK9 inhibitors significantly decreased the activation of pERK1/2 and p4EBP1(mTOR) with a concomitant decrease in MYC and NME1 at the protein level. We next explored these inhibitors for treating murine RS in vivo. We established murine RS cells by engrafting RS cells into NSG mice and then treated RS mice by orally given vehicle (n = 13); AZ5576 (60mg/kg) (n = 13); TTFA (25mg/kg) (n = 4); and AZ + TTFA (60 and 25mg/kg) (n = 5). AZ5576 and TTFA single treatment significantly prolonged the survival of RS mice (Log-rank test, p < 0.0001) with a reduction in total spleen weight (p=0.0007). AZ+TTFA combination had synergy in increasing the survival of RS mice by 10 days (p=0.0003) compared to a single treatment. AZ+TTFA treated murine RS cells showed reduced MYC, NME1, and p4E-BP1 protein expression, confirming the effectiveness of targeting of Mga-Myc-Nme1 axis. In summary, our murine model reveals that MGA/MYC/NME1 axis drives RS through increased mitochondrial OXPHOS and ROS accumulation. Moreover, targeting this axis provides therapeutic benefits for RS, highlighting this pathway as a novel potential target for RS treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

ATM serine/threonine kinase germline mutations in chronic lymphocytic leukaemia come in different flavours.

ATM serine/threonine kinase germline mutations in chronic lymphocytic leukaemia come in different flavours.

The spectrum of subclonal TP53 mutations in chronic lymphocytic leukemia: A next generation sequencing retrospective study.

Chronic lymphocytic leukemia (CLL) is a hematological disorder with complex clinical and biological behavior. TP53 mutational status and cytogenetic assessment of the deletion of the corresponding locus (17p13.1) are considered the most relevant biomarkers associated with pharmaco-predictive response, chemo-refractoriness, and worse prognosis in CLL patients. The implementation of Next Generation Sequencing (NGS) methodologies in the clinical laboratory allows for comprehensively analyzing the TP53 gene and detecting mutations with allele frequencies ≤10%, that is, "subclonal mutations". We retrospectively studied TP53 gene mutational status by NGS in 220 samples from 171 CLL patients. TP53 mutations were found in 60/220 (27.3%) samples and 47/171 (27.5%) patients. Interestingly, subclonal mutations could be detected in 31/60 samples (51.7%) corresponding to 25 patients (25/47, 53.2%). We identified 44 distinct subclonal TP53 mutations clustered in the central DNA-binding domain of p53 protein (exons 5-8, codons 133-286). Missense mutations were predominant (>80%), whereas indels, nonsense, and splice site variants were less represented. All subclonal TP53 variants but one [p.(Pro191fs)] were already described in NCI and/or Seshat databases as "damaging" and/or "probably damaging" mutations (38/44, 86% and 6/44, 14%, respectively). Longitudinal samples were available for 37 patients. Almost half of them displayed at least one TP53 mutant subclone, which could be alone (4/16, 25%) or concomitant with other TP53 mutant clonal ones (12/16, 75%); different patterns of mutational dynamics overtimes were documented. In conclusion, utilization of NGS in our "real-life" cohort of CLL patients demonstrated an elevated frequency of subclonal TP53 mutations. This finding indicates the need for precisely identifying these mutations during disease since the clones carrying them may become predominant and be responsible for therapy failures.

High-resolution Melting Analysis for NOTCH1 c.7541-7542delCT Mutation in Chronic Lymphocytic Leukemia: Prognostic Significance in Egyptian Patients.

The present study aimed to detect the prevalence of NOTCH1 c.7541-7542delCT mutation in Egyptian CLL patients using HRM assay and to assess its relation to patients' survival. The study included 50 newly diagnosed treatment-naïve CLL patients and 50 age and sex matched healthy controls. NOTCH1 c.7541-7542delCT mutation was detected using High-resolution melting (HRM) assay and direct Sanger sequencing. Outcome parameters included progression free survival (PFS) and overall survival (OS). NOTCH1 c.7541-7542delCT mutation was detected in 5 (10.0%) of CLL patients. No controls had NOTCH1 c.7541-7542delCT mutation. Similar results were obtained by direct Sanger sequencing yielding a sensitivity and specificity of 100.0% for HRM in detection of NOTCH1 c.7541-7542delCT mutation in the studied patients. In univariate analysis, predictors of OS included Trisomy 12, high LDH, presence of NOTCH1 c.7541-7542delCT mutation and lack of CR. In multivariate analysis, only lack of CR was found as a significant predictor of OS. HRM analysis is a sensitive method for detection of NOTCH1 c.7541-7542delCT mutation in CLL patients. This mutation may be linked to poor disease prognosis.

Genetic Features and TP53 Mutations in Chronic Lymphocytic Leukemia

Genetic Features and TP53 Mutations in Chronic Lymphocytic Leukemia

MGA deletion Leads to Richter's Transformation Via NME1

MGA deletion Leads to Richter's Transformation Via NME1

NOTCH1 Mutation in CLL Enhances Cell Cycle G1/S Transition through Specific Cyclin Overexpression: Preclinical Ground for CDK4/6 Targeted Inhibition

Introduction: The partially understood biological consequences of the NOTCH1 acquired lesion, seems to be distinctive enough among chronic lymphocytic leukemia (CLL) patients, as clinical studies have repeatedly found specific features: intermediate prognosis, anti-CD20 poorer responses, and a higher frequency of trisomy 12 and Richter transformation. Though located in a different domain, the activating nature of NOTCH1 mutation in T lymphoblastic leukemia relies on cell cycle regulators. In fact, pivotal studies, from the pre-next generation sequencing era, showed dysregulation of cyclins-gene expression, as driver of the unique CLL features. Thus, our goal was to revisit the cell cycle in CLL, but focusing now in the NOTCH1 mutated subset (NOTCH1MUT), hypothesizing that biological differences versus wild type cases (NOTCH1WT) would explain the clinical ones, and exploiting potential differences with targeted molecules in vitro.Methods: From 2010 to 2019, presentation bone marrow aspirates or blood samples DNA was collected during the diagnostic workout from 378 CLL patients, all of them annotated by next generation sequencing. G 0/early-G 1 effectors gene expression was measured by RT-qPCR in negatively immunoselected circulating CLL cells. A siRNA approach was selected for silencing by electroporation 7 NOTCH1WT and 2 NOTCH1MUT cases. Cell cycle and apoptosis flow cytometry assays were performed on cultured fresh primary cells from n? NOTCH1MUT and 4 NOTCH1WT cases, before and after exposure to different concentrations of palbociclib, a CDK4/6 inhibitor.Results: We found that 37/378 (9.8%) of patients harbored a NOTCH1 mutation. NOTCH1MUTcases presented with higher lymphocyte counts [NOTCH1MUT 17.2 x10 9/L vs. NOTCH1WT 9.7 x10 9/L; p=0.042], trisomy 12 (35.1% vs. 11.4%; p<0.001) and a higher frequency of an unmutated IGHV status (70% vs. 21%; p<0.001). Of note, NOTCH1MUT patients had poorer responses to anti-CD20 based schemes than NOTCH1WT patients (35.7 vs. 69.8% complete response; p). We found that NOTCH1MUT cases showed a relevant increase of 38-fold change (FC) for CCND3, 27-FC for CDK4 and CCND2, 11-FC for CCND1 and 9-FC for CDK6 gene expression in negatively immunoselected circulating CLL cells at diagnosis. In addition, NOTCH1MUT cases displayed a statistically significant higher percentage of cells in the S phase than the wild type cases (21% vs. 1%, p=0.004). Though significance was not met, NOTCH1MUT cases showed a higher percentage of events within G 2-M (28% vs. 26 %). Next, we incorporated the flow cytometry assay to in vitro palbociclib treated CLL cells from 3 NOTCH1MUT and 4 NOTCH1WT cases. Five days after culture stimulation, cells were exposed to 38 and 76 μM (dose range for reaching maximum CLL cells sensitivity plateau) of the drug for 48 hours. As stated above, NOTCH1MUT cases were characterized by a much higher proportion of cells in S phase at baseline (21%), which was reduced in a dose dependent manner to an 8% and a 6% after exposure to palbociclib, respectively. The standard 48-72 hours drug assay may not be the most suitable for slow growth tumors as CLL and, in particular, for testing cell cycle inhibitors. Thus, we designed an assay for two cell cycles based on the average population doubling time of the primary cell culture experiments (0.6 in 72 hours), and using the mean steady state plasma concentration of palbociclib achieved clinically: 1 μM. After 120 hours, the baseline 15% of cells in S phase was reduced to an 0.64% after exposure to palbociclib 1 μM in NOTCH1MUT cases and a 1.8x-increase in the percentage dead cells was noted, compared with NOTCH1WT cases.Conclusions: Compared with NOTCH1WT CLL cases, we describe an overexpression of effectors of early phase in NOTCH1MUT. This profile made NOTCH1MU cells more suited to enter and traverse through the cell cycle and could explain, in part, the proliferative clinical-biological features of this subset of patients and opening a window for exploiting therapeutically these differences. Ours experiments in vitro with palbociclib sets the ground for the clinical research. [Display omitted] DisclosuresJerez: BMS: Consultancy; Novartis: Consultancy; GILEAD: Research Funding.

Prognostic significance of ATM mutations in chronic lymphocytic leukemia: A meta-analysis

BackgroundThe ATM protein acts as an essential part of the signal transduction pathway upstream of p53 which activates following induction of DNA double-strand breaks (DSBs) and leads to transcriptional proapoptotic genes activation that synchronizes DNA repair. AimSeveral studies have assessed the relationship between ATM mutations and the clinical prognosis in patients with chronic lymphocytic leukemia (CLL). However, its prognostic value has not yet been fully clarified. Hence, we aimed this meta-analysis to investigate the prognostic effect of ATM mutations in patients with CLL. MethodThe selected clinical studies were extracted from various electronic databases such as PubMed, EMBASE, the Cochrane Library, and Web of Science. In our meta-analysis, Hazard Ratio (HRs) and 95 % confidence interval (CI) for overall survival (OS) were chosen to estimate the prognostic impact of ATM mutations and to compare ATM mutations to those with wild-type. ResultsA total of 1299 patients from seven studies were collected. The pooled HRs for OS recommended that patients with CLL had a poorer prognosis HR = 1.24 (95 % CI: 0.97–1.59). The incidence of ATM mutations was found 15.8 % in patients with CLL. Begg’s and Egger’s tests did not show any significant bias between studies. ConclusionIn conclusion, this meta-analysis indicated that ATM mutations were significantly associated with adverse prognostic effect in patients with CLL. However, a randomized controlled prospective study with a large number of patients with different types of ATM mutations is required to assert these results.

Standardized Targeted Sequencing of 29 Genes with Recurrent Mutations in Chronic Lymphocytic Leukemia (CLL) Helps to Better Define Clinical Correlates and Genomic Hotspots

Standardized Targeted Sequencing of 29 Genes with Recurrent Mutations in Chronic Lymphocytic Leukemia (CLL) Helps to Better Define Clinical Correlates and Genomic Hotspots

Rituximab Activates NOTCH1 Signaling in CLL Cells and Induces Changes in the Cytokine Repertoire Favoring a Tumor-Protective Microenvironment

IntroductionThe addition of the anti-CD20 monoclonal antibody (mAb) rituximab to chemotherapy improves responses in almost all chronic lymphocytic leukemia (CLL) patients, with the exception of those having NOTCH1 mutations. The reasons for this are largely unknown.NOTCH1 is a cell surface receptor releasing its intracellular domain (NICD) upon activation. The NICD is a transcription factor and NOTCH1 mutations in CLL lead to longer lasting transcription factor activity.We studied the effects of rituximab on NOTCH1 receptor activity and aimed to identify mechanisms of resistance towards chemoimmunotherapy based on NOTCH1 signaling.Material & MethodsFreshly isolated peripheral blood mononuclear cells from CLL patients attending St. Bartholomew's Hospital, London were enriched for CD19 positive cells by negative selection using EDTA free buffer. Cells were treated with mAbs for various time periods. Whole protein lysates were obtained after 15, 30 and 60 min of mAb treatment; RNA was isolated after 150 min. NICD was semi-quantitatively assessed by western blot and NICD target gene expression, such as expression of HES1, was quantified by TaqMan® probe-based quantitative PCR. Global gene expression profiling was done using Affymetrix® Human Gene ST 2.0 Arrays on 3 biological replicates of treated and non-treated samples each deriving from the same patient.ResultsCLL cells freshly isolated from peripheral blood showed low NOTCH1 signaling activity. Following in-vitro treatment of cells with rituximab for 15, 30 or 60 min we observed NOTCH1 receptor activation with NICD release in a time-dependent manner, regardless of NOTCH1 mutation status. We used appropriate controls to distinguish whether NOTCH1 activation was associated with CD20 engagement and/or Fcγ-receptor (FcγR) IIB binding. Trastuzumab was used as an isotype control and led to stronger NOTCH1 activation than F(ab')2 fragments of rituximab, suggesting that NICD release mainly occurs upon engagement of FcγRIIB.In line with this, in-vitro treatment of cells with rituximab was followed by up-regulated expression of NICD target genes such as HES1 . Global gene expression analysis when maximum HES1 gene expression was observed revealed 139 up-regulated and 124 down-regulated probesets in an analysis comparing rituximab treated with non-treated samples (fold-change ≥1.5; p-value ≤0.05) with the majority being non-coding RNAs. Only 28 differentially regulated probesets were actually coding for known genes. Gene set enrichment analysis against the curated genesets of the MSigDB collection (C2) identified 9 enriched genesets (false discovery rate <25%), mainly related to cytokine secretion.The two most up-regulated genes were CCL2 and CCL4 with fold-changes of 6.5 and 3.2, respectively (p<0.001). The gene locus of CCL2 was previously identified as a direct NICD target and CCL2 and CCL4 were both shown to be produced by CLL cells. Numerous studies have linked CCL2 and CCL4 with the recruitment of tumor-protective immune cells such as tumor associated macrophages and T-cell subsets thereby changing the composition of the tumor microenvironment.While wild-type NICD induced only short-lived gene expression changes after transient NOTCH1 activation, mutant NICD elicited more sustained changes. Since NOTCH1 activation following rituximab binding is likely to be temporary, we hypothesise that expression changes of cytokines such as CCL2 and CCL4 last long enough only in NOTCH1 mutant CLL to induce profound changes in the tumor microenvironment.ConclusionIn summary, our data shows for the first time that rituximab binding to CLL cells can activate NOTCH1 signaling and influence the cells' transcriptome. We believe that NOTCH1 induced changes to the cytokine repertoire contribute to the formation of a tumor-protective microenvironment and that the extent of this is dependent on the durability of NICD's transcription factor activity, which is prolonged in NOTCH1 mutant CLL.In ongoing studies we are testing the effects of ofatumumab and obinutuzumab on NOTCH1 signaling and we are using sequential samples taken before and after anti-CD20 mAb infusion to define the extent to which rituximab can influence NOTCH1 signaling in-vivo. We also aim to characterise the cellular composition of the CLL microenvironment with regard to NOTCH1 mutation status and NOTCH1 activity. DisclosuresGribben:Kite: Honoraria; Karyopharm: Honoraria; Acerta: Honoraria; Pharmacyclics: Honoraria; Celgene: Honoraria; TG Therapeutics: Honoraria; Abbvie: Honoraria; Janssen: Honoraria; Genentech/Roche: Honoraria.

BRAF and BIRC3 Mutations Stratify a Poor Prognostic Subgroup in FCR Treated Chronic Lymphocytic Leukemia

Abstract Background. The current shift of therapy of chronic lymphocytic leukemia (CLL) towards novel targeted agents mandates the recognition of molecular predictors to identify patients who can still benefit from chemoimmunotherapy and those who should instead be considered for novel targeted agents upfront. In the case of FCR (fludarabine, cyclophosphamide, rituximab), the IGHV mutation status and FISH karyotype stratify low-risk patients carrying mutated IGHV genes and devoid of both del11q and del17p who maximally benefit from such treatment; intermediate-risk patients harboring unmutated IGHV genes and/or del11q in the absence of del17p are a case mix of good and poor responders to FCR; while high-risk patients harboring del17p are unsuitable for chemoimmunotherapy. This model fails to consider the impact of recurrent CLL mutations of potential prognostic relevance, which may be improved by their inclusion. Purpose. We aimed at refining the genetic-based stratification of FCR-treated CLL patients by integrating the mutational profile in a prognostic model together with the IGHV mutation status and FISH karyotype. Methods. A multicenter cohort of 173 (162 with complete molecular data) untreated CLL receiving first-line therapy with FCR in the real-life clinical practice was evaluated by target resequencing. Tumor genomic DNA collected at the time of treatment was analyzed for mutations in the coding exons plus splice sites of CLL cancer driver genes (n=23). Deep next-generation-sequencing (NGS) of the gene panel was performed on the Illumina MiSeq platform (coverage >2000x in >90% of the target). Non-synonymous mutations represented in >10% of tumor allele were called by using VarScan2, and a stringent bioinformatic pipeline was developed to protect against the false call of polymorphisms and sequencing errors. Medical statistics was performed using SPSS version 24.0 and R version 3.3.2. Results. The cohort characteristics and mutational profile were consistent with those reported in CLL receiving FCR as initial treatment. After a median follow-up of 7.2 years, 114 patients progressed, accounting for a median PFS of 4.5 years. Among patients categorized as low-risk by IGHV and FISH status, gene mutations associated with poor prognosis were virtually absent (Fig. 1A). By univariate analysis, none of the cancer driver gene mutations significantly associated with PFS among low-risk patients. Consistently, by recursive partitioning, the proportion of low-risk patients failing FCR early was not explained by the co-occurrence of a cancer driver gene mutation. Among patients categorized as intermediate-risk by IGHV and FISH status, mutations of BIRC3 (HR: 6.452; 95% CI 2.467-16.875; p Download : Download high-res image (256KB) Download : Download full-size image Disclosures Forconi: AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Infinity: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Janssen-Cilag: Speakers Bureau. Zaja: Novartis: Honoraria, Research Funding; Janssem: Honoraria; Roche: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Abbvie: Honoraria; Takeda: Honoraria; Gilead: Honoraria. Rigolin: Celgene: Honoraria, Other: Advisory Board; Mundipharma: Other: Advisory Board; Gilead: Honoraria. Coscia: Abbvie: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Tedeschi: AbbVie: Consultancy; Gilead: Consultancy, Other: travel expenses; Janssen: Consultancy, Other: travel expenses. Laurenti: Roche: Other: Advisory Board; Gilead: Other: Advisory Board; Janssen: Other: Advisory Board; Abbvie: Other: Advisory Board. Cuneo: Gilead: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Abbvie: Honoraria, Other: Advisory Board; Roche: Honoraria, Other: Advisory Board. Foa: AbbVie: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Sandoz: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau. Gaidano: Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

Challenges with Approved Targeted Therapies against Recurrent Mutations in CLL: A Place for New Actionable Targets.

Simple SummaryChronic lymphocytic leukemia harbors a high degree of genetic variability and interpatient heterogeneity. Some of the genetic alterations have an impact on the disease’s prognosis and evolution, but few data exist about the response to new approved targeted therapies in patients carrying recurrent mutations other than TP53. In this review, we present the knowledge about the impact of these new genetic alterations in the treatment response together with the possibility to use new actionable targets.Chronic lymphocytic leukemia (CLL) is characterized by a high degree of genetic variability and interpatient heterogeneity. In the last decade, novel alterations have been described. Some of them impact on the prognosis and evolution of patients. The approval of BTK inhibitors, PI3K inhibitors and Bcl-2 inhibitors has drastically changed the treatment of patients with CLL. The effect of these new targeted therapies has been widely analyzed in TP53-mutated cases, but few data exist about the response of patients carrying other recurrent mutations. In this review, we describe the biological pathways recurrently altered in CLL that might have an impact on the response to these new therapies together with the possibility to use new actionable targets to optimize treatment responses.