Mutations Detected in Real World Clinical Sequencing during BTK Inhibitor Treatment in CLL.

We retrospectively analyzed 609 chronic lymphocytic leukemia (CLL) patients treated with BTK inhibitors (BTKis) at Dana-Farber Cancer Institute from 2014 to 2022. Among them, 85 underwent next-generation sequencing (NGS) during or after BTKi therapy (ibrutinib, 64; acalabrutinib, 13; pirtobrutinib, 7; vecabrutinib, 1). Patients with NGS at progression (N=36, PD group) showed more 17p deletion, complex karyotype, and previous treatments including BTKi, compared to ongoing responders (N=49, NP group). 216 variants were found in 57 genes across both groups, with more variants in the PD group (158 variants, 70.3% pathogenic, P<0.001). The PD group had a higher incidence of pathogenic variants (70.3%, P<0.001), including 32 BTK(BTK C481S/F/R/Y, L528W, and T474I/L) and 4 PLCG2mutations. Notably, a high VAF L528W mutation was found in a first line ibrutinib-resistant patient. TP53, SF3B1, and NOTCH2mutations were also significantly more prevalent in the PD group (P<0.01, P<0.05, P<0.05). Additionally, MAPK pathway gene mutations trended more common and had higher VAFs in the PD group (P=0.041). T474 mutations were found in 4 of 6 patients progressing on pirtobrutinib, and BTK L528W mutation can arise with both covalent and non-covalent BTKi therapy. These results also suggest that RAS/RAF/MAPK pathway mutations may contribute to BTKi resistance.

The influence of sampling method and season on modeling of selenium into coldwater fish and implications on tissue-based water quality benchmarks.

Selenium (Se) contamination of aquatic ecosystems has led to the local extirpation of some Se-sensitive fish species. Although Se exposure occurs primarily via diet, considerable uncertainty lies in modeling Se transfer and bioaccumulation from sediment, detritus, and/or periphyton through benthic macroinvertebrates (BMI) to fish. Here we estimated Se concentrations in four coldwater fish species (northern pike, white sucker, lake whitefish, and ninespine stickleback) inhabiting boreal lakes downstream from a uranium mill in northern Canada. In addition, we evaluated the potential effects of BMI and periphyton sampling methods (artificial substrates vs. grab samples), seasons (summer vs. winter), and models (USEPA vs. Assessment of the Dispersion and Effects of Parameter Transport) on the estimated Se concentrations in fish tissue. Results were compared with site-specific benchmarks and observed Se concentrations in resident fish. In summer 2019, periphyton and BMI were sampled at 10 sampling stations (two in Vulture Lake and eight in McClean Lake) using artificial substrates (n = 4) and sediment grab samples (n = 3). In winter 2021, samples were collected in McClean Lake (n = 3) through ice holes using a sediment grab sampler. Estimated Se concentrations in fish tissue depended on the surface sediment or periphyton Se concentrations used in the models. At Vulture Lake, Se concentrations in northern pike muscle estimated using the grab sample data (17.3 ± 11.5 µg/g DW), but not the artificial substrates (34.5 ± 1.2 µg/g DW), were comparable with the observed mean concentration (19.0 ± 1.4 µg/g DW) in this species. At McClean Lake, Se body burdens in forage fish estimated using data from both sampling methods were comparable with measured data. Significantly lower mean whole-body Se concentrations were estimated for all fish species in winter (1.0 ± 0.3 µg/g DW) relative to summer (4.8 ± 1.6 µg/g DW). Further investigation is necessary to understand how potential seasonal shifts in dietary Se exposure relate to fish reproduction and early life stages. Integr Environ Assess Manag 2023;00:1-15. © 2023 SETAC.

Genetic events associated with venetoclax resistance in CLL identified by whole-exome sequencing of patient samples.

Although BCL2 mutations are reported as later occurring events leading to venetoclax resistance, many other mechanisms of progression have been reported though remain poorly understood. Here, we analyze longitudinal tumor samples from 11 patients with disease progression while receiving venetoclax to characterize the clonal evolution of resistance. All patients tested showed increased in vitro resistance to venetoclax at the posttreatment time point. We found the previously described acquired BCL2-G101V mutation in only 4 of 11 patients, with 2 patients showing a very low variant allele fraction (0.03%-4.68%). Whole-exome sequencing revealed acquired loss(8p) in 4 of 11 patients, of which 2 patients also had gain (1q21.2-21.3) in the same cells affecting the MCL1 gene. In vitro experiments showed that CLL cells from the 4 patients with loss(8p) were more resistant to venetoclax than cells from those without it, with the cells from 2 patients also carrying gain (1q21.2-21.3) showing increased sensitivity to MCL1 inhibition. Progression samples with gain (1q21.2-21.3) were more susceptible to the combination of MCL1 inhibitor and venetoclax. Differential gene expression analysis comparing bulk RNA sequencing data from pretreatment and progression time points of all patients showed upregulation of proliferation, B-cell receptor (BCR), and NF-κB gene sets including MAPK genes. Cells from progression time points demonstrated upregulation of surface immunoglobulin M and higher pERK levels compared with those from the preprogression time point, suggesting an upregulation of BCR signaling that activates the MAPK pathway. Overall, our data suggest several mechanisms of acquired resistance to venetoclax in CLL that could pave the way for rationally designed combination treatments for patients with venetoclax-resistant CLL.

METTL3-Mediated m6A Modification Controls Splicing Factor Abundance and Contributes to Aggressive CLL.

RNA splicing dysregulation underlies the onset and progression of cancers. In chronic lymphocytic leukemia (CLL), spliceosome mutations leading to aberrant splicing occur in ∼20% of patients. However, the mechanism for splicing defects in spliceosome-unmutated CLL cases remains elusive. Through an integrative transcriptomic and proteomic analysis, we discover that proteins involved in RNA splicing are posttranscriptionally upregulated in CLL cells, resulting in splicing dysregulation. The abundance of splicing complexes is an independent risk factor for poor prognosis. Moreover, increased splicing factor expression is highly correlated with the abundance of METTL3, an RNA methyltransferase that deposits N6-methyladenosine (m6A) on mRNA. METTL3 is essential for cell growth in vitro and in vivo and controls splicing factor protein expression in a methyltransferase-dependent manner through m6A modification-mediated ribosome recycling and decoding. Our results uncover METTL3-mediated m6A modification as a novel regulatory axis in driving splicing dysregulation and contributing to aggressive CLL. SIGNIFICANCE: METTL3 controls widespread splicing factor abundance via translational control of m6A-modified mRNA, contributes to RNA splicing dysregulation and disease progression in CLL, and serves as a potential therapeutic target in aggressive CLL. See related commentary by Janin and Esteller, p. 176. This article is highlighted in the In This Issue feature, p. 171.

Circulating Th17 T cells at treatment onset predict autoimmune toxicity of PI3Kδ inhibitors.

PI3Kδ inhibitors are approved for the therapy of B cell malignancies, but their clinical use has been limited by unpredictable autoimmune toxicity, despite promising efficacy and evidence that toxicity is associated with improved clinical outcomes. Prior phenotypic evaluation by CyTOF has identified increases in activated CD8 T cells with activation of Th17 T cells, as well as decreases in Tregs, particularly in patients with toxicity. Here we sought to further understand the effects of idelalisib and duvelisib in vitro, and demonstrate that both idelalisib and duvelisib can inhibit T cell proliferation as well as Th1 and Treg differentiation in vitro, while promoting Th2 and Th17 differentiation. We further demonstrate directly using intracellular flow cytometry that autoimmune toxicity in patients is associated with higher absolute numbers of CD4 and CD8 T cells with Th17 differentiation in peripheral blood prior to therapy, and that gastrointestinal tissues from patients with active autoimmune complications of PI3Kδ inhibitors show infiltration with Th17+ T cells. These same tissues show depletion of Tregs as compared to CLL patients without toxicity, suggesting that loss of Tregs may be permissive for Th17 activation to lead to autoimmune toxicity. Clinical trials to restore this balance are warranted.

Evolutionary history of transformation from chronic lymphocytic leukemia to Richter syndrome.

Richter syndrome (RS) arising from chronic lymphocytic leukemia (CLL) exemplifies an aggressive malignancy that develops from an indolent neoplasm. To decipher the genetics underlying this transformation, we computationally deconvoluted admixtures of CLL and RS cells from 52 patients with RS, evaluating paired CLL-RS whole-exome sequencing data. We discovered RS-specific somatic driver mutations (including IRF2BP2, SRSF1, B2M, DNMT3A and CCND3), recurrent copy-number alterations beyond del(9p21)(CDKN2A/B), whole-genome duplication and chromothripsis, which were confirmed in 45 independent RS cases and in an external set of RS whole genomes. Through unsupervised clustering, clonally related RS was largely distinct from diffuse large B cell lymphoma. We distinguished pathways that were dysregulated in RS versus CLL, and detected clonal evolution of transformation at single-cell resolution, identifying intermediate cell states. Our study defines distinct molecular subtypes of RS and highlights cell-free DNA analysis as a potential tool for early diagnosis and monitoring.

A phase 1b study of ibrutinib in combination with obinutuzumab in patients with relapsed or refractory chronic lymphocytic leukemia.

This study investigated ibrutinib plus obinutuzumab in relapsed/refractory CLL, evaluating tolerability of 3 sequencing regimens as well as overall safety and efficacy. Fifty-two patients were initially randomized 1:1:1 to receive either obinutuzumab 1 month before ibrutinib initiation, ibrutinib 1 month prior to obinutuzumab initiation, or to start both drugs concomitantly. Higher rates of infusion-related reactions were observed with the first sequence, and only the latter 2 cohorts were expanded. Grade 4 hematologic toxicity was uncommon, and notable all-grade non-hematologic toxicities included bruising (58%), hypertension (46%), arthralgia (38%), diarrhea (37%), transaminitis (35%), atrial fibrillation (21%), and serious infection (17%). Best overall response rate was 96% (including 40% CR and 56% PR). Best rates of undetectable minimal residual disease in peripheral blood and bone marrow were 27% and 19%, respectively. With a median follow-up of 41.5 months, four-year progression-free and overall survival rates are 74% and 93%, respectively. Correlative studies demonstrated that serum CCL4 and CXCL13 levels were associated with clinical response, and BH3 profiling revealed increased BCL-2 and BCL-xL dependence in CLL cells from patients on treatment. Overall, ibrutinib plus obinutuzumab was highly active, with a manageable safety profile, supporting further investigation of this type of approach in relapsed/refractory CLL.

Pirtobrutinib targets BTK C481S in ibrutinib-resistant CLL but second-site BTK mutations lead to resistance.

Covalent inhibitors of Bruton tyrosine kinase (BTK) have transformed the therapy of chronic lymphocytic leukemia (CLL), but continuous therapy has been complicated by the development of resistance. The most common resistance mechanism in patients whose disease progresses on covalent BTK inhibitors (BTKis) is a mutation in the BTK 481 cysteine residue to which the inhibitors bind covalently. Pirtobrutinib is a highly selective, noncovalent BTKi with substantial clinical activity in patients whose disease has progressed on covalent BTKi, regardless of BTK mutation status. Using in vitro ibrutinib-resistant models and cells from patients with CLL, we show that pirtobrutinib potently inhibits BTK-mediated functions including B-cell receptor (BCR) signaling, cell viability, and CCL3/CCL4 chemokine production in both BTK wild-type and C481S mutant CLL cells. We demonstrate that primary CLL cells from responding patients on the pirtobrutinib trial show reduced BCR signaling, cell survival, and CCL3/CCL4 chemokine secretion. At time of progression, these primary CLL cells show increasing resistance to pirtobrutinib in signaling inhibition, cell viability, and cytokine production. We employed longitudinal whole-exome sequencing on 2 patients whose disease progressed on pirtobrutinib and identified selection of alternative-site BTK mutations, providing clinical evidence that secondary BTK mutations lead to resistance to noncovalent BTKis.

Rare Germline ATM Variants Influence the Development of Chronic Lymphocytic Leukemia.

PURPOSE: Germline missense variants of unknown significance in cancer-related genes are increasingly being identified with the expanding use of next-generation sequencing. The ataxia telangiectasia-mutated (ATM) gene on chromosome 11 has more than 1,000 germline missense variants of unknown significance and is a tumor suppressor. We aimed to determine if rare germline ATM variants are more frequent in chronic lymphocytic leukemia (CLL) compared with other hematologic malignancies and if they influence the clinical characteristics of CLL. METHODS: We identified 3,128 patients (including 825 patients with CLL) in our hematologic malignancy clinic who had received clinical-grade sequencing of the entire coding region of ATM. We ascertained the comparative frequencies of germline ATM variants in categories of hematologic neoplasms, and, in patients with CLL, we determined whether these variants affected CLL-associated characteristics such as somatic 11q deletion. RESULTS: Rare germline ATM variants are present in 24% of patients with CLL, significantly greater than that in patients with other lymphoid malignancies (16% prevalence), myeloid disease (15%), or no hematologic neoplasm (14%). Patients with CLL with germline ATM variants are younger at diagnosis and twice as likely to have 11q deletion. The ATM variant p.L2307F is present in 3% of patients with CLL, is associated with a three-fold increase in rates of somatic 11q deletion, and is a hypomorph in cell-based assays. CONCLUSION: Germline ATM variants cluster within CLL and affect the phenotype of CLL that develops, implying that some of these variants (such as ATM p.L2307F) have functional significance and should not be ignored. Further studies are needed to determine whether these variants affect the response to therapy or account for some of the inherited risk of CLL.

Molecular map of chronic lymphocytic leukemia and its impact on outcome.

Recent advances in cancer characterization have consistently revealed marked heterogeneity, impeding the completion of integrated molecular and clinical maps for each malignancy. Here, we focus on chronic lymphocytic leukemia (CLL), a B cell neoplasm with variable natural history that is conventionally categorized into two subtypes distinguished by extent of somatic mutations in the heavy-chain variable region of immunoglobulin genes (IGHV). To build the 'CLL map,' we integrated genomic, transcriptomic and epigenomic data from 1,148 patients. We identified 202 candidate genetic drivers of CLL (109 new) and refined the characterization of IGHV subtypes, which revealed distinct genomic landscapes and leukemogenic trajectories. Discovery of new gene expression subtypes further subcategorized this neoplasm and proved to be independent prognostic factors. Clinical outcomes were associated with a combination of genetic, epigenetic and gene expression features, further advancing our prognostic paradigm. Overall, this work reveals fresh insights into CLL oncogenesis and prognostication.

Functional Testing to Characterize and Stratify PI3K Inhibitor Responses in Chronic Lymphocytic Leukemia.

PURPOSE: PI3K inhibitors (PI3Ki) are approved for relapsed chronic lymphocytic leukemia (CLL). Although patients may show an initial response to these therapies, development of treatment intolerance or resistance remain clinical challenges. To overcome these, prediction of individual treatment responses based on actionable biomarkers is needed. Here, we characterized the activity and cellular effects of 10 PI3Ki and investigated whether functional analyses can identify treatment vulnerabilities in PI3Ki-refractory/intolerant CLL and stratify responders to PI3Ki. EXPERIMENTAL DESIGN: Peripheral blood mononuclear cell samples (n = 51 in total) from treatment-naïve and PI3Ki-treated patients with CLL were studied. Cells were profiled against 10 PI3Ki and the Bcl-2 antagonist venetoclax. Cell signaling and immune phenotypes were analyzed by flow cytometry. Cell viability was monitored by detection of cleaved caspase-3 and the CellTiter-Glo assay. RESULTS: pan-PI3Kis were most effective at inhibiting PI3K signaling and cell viability, and showed activity in CLL cells from both treatment-naïve and idelalisib-refractory/intolerant patients. CLL cells from idelalisib-refractory/intolerant patients showed overall reduced protein phosphorylation levels. The pan-PI3Ki copanlisib, but not the p110δ inhibitor idelalisib, inhibited PI3K signaling in CD4+ and CD8+ T cells in addition to CD19+ B cells, but did not significantly affect T-cell numbers. Combination treatment with a PI3Ki and venetoclax resulted in synergistic induction of apoptosis. Analysis of drug sensitivities to 73 drug combinations and profiling of 31 proteins stratified responders to idelalisib and umbralisib, respectively. CONCLUSIONS: Our findings suggest novel treatment vulnerabilities in idelalisib-refractory/intolerant CLL, and indicate that ex vivo functional profiling may stratify PI3Ki responders.

Idelalisib activates AKT via increased recruitment of PI3Kδ/PI3Kß to BCR signalosome while reducing PDK1 in post-therapy CLL cells.

Idelalisib targets PI3Kδ in the BCR pathway generating only a partial response in CLL patients, indicating that the leukemic cells may have evolved escape signals. Indeed, we detected increased activation of AKT accompanied by upregulation of MYC/BCL2 in post-therapy CLL cells from patients treated with idelalisib/ofatumumab. To unravel the mechanism of increased AKT-activation, we studied the impact of idelalisib on a CLL-derived cell line, MEC1, as a model. After an initial inhibition, AKT-activation level was restored in idelalisib-treated MEC1 cells in a time-dependent manner. As BCAP (B-cell adaptor for PI3K) and CD19 recruit PI3Kδ to activate AKT upon BCR-stimulation, we examined if idelalisib-treatment altered PI3Kδ-recruitment. Immunoprecipitation of BCAP/CD19 from idelalisib-treated MEC1 cells showed increased recruitment of PI3Kδ in association with PI3Kß, but not PI3Kα or PI3Kγ and that, targeting both PI3Kδ with PI3Kß inhibited AKT-reactivation. We detected similar, patient-specific recruitment pattern of PI3K-isoforms by BCAP/CD19 in post-idelalisib CLL cells with increased AKT-activation. Interestingly, a stronger inhibitory effect of idelalisib on P-AKT (T308) than S473 was discernible in idelalisib-treated cells despite increased recruitment of PI3Kδ/PI3Kß and accumulation of phosphatidylinositol-3,4,5-triphosphate; which could be attributed to reduced PDK1 activity. Thus, administration of isoform-specific inhibitors may prove more effective strategy for treating CLL patients.

Idelalisib reduces regulatory T cells and activates T helper 17 cell differentiation in relapsed refractory patients with chronic lymphocytic leukaemia.

Phosphatidylinositol 3 kinase (PI3K) inhibitors such as idelalisib have been associated with potentially severe autoimmune toxicity. In the present study, we demonstrate that relapsed refractory patients with chronic lymphocytic leukaemia treated with idelalisib rituximab on the phase III registration trial show uniform decrease in regulatory T cells (Tregs) and increase in CD8 T cells with treatment. Patients who do not develop toxicity show enrichment for T cells expressing multiple chemokine receptors, while those who do develop toxicity have an activated CD8 T cell population with T helper 17 cell differentiation at baseline, which then increases, leading to an increased CD8:Treg ratio that likely triggers autoimmune toxicity.

A T cell inflammatory phenotype is associated with autoimmune toxicity of the PI3K inhibitor duvelisib in chronic lymphocytic leukemia.

Several PI3Kδ inhibitors are approved for the therapy of B cell malignancies, but their clinical use has been limited by unpredictable autoimmune toxicity. We have recently reported promising efficacy results in treating chronic lymphocytic leukemia (CLL) patients with combination therapy with the PI3Kδγ inhibitor duvelisib and fludarabine cyclophosphamide rituximab (FCR) chemoimmunotherapy, but approximately one-third of patients develop autoimmune toxicity. We show here that duvelisib FCR treatment in an upfront setting modulates both CD4 and CD8 T cell subsets as well as pro-inflammatory cytokines. Decreases in naive and central memory CD4 T cells and naive CD8 T cells occur with treatment, while activated CD8 T cells, granzyme positive Tregs, and Th17 CD4 and CD8 T cells all increase with treatment, particularly in patients with toxicity. Cytokines associated with Th17 activation (IL-17A and IL-21) are also relatively elevated in patients with toxicity. The only CLL feature associated with toxicity was increased priming for apoptosis at baseline, with a significant decrease during the first week of duvelisib. We conclude that an increase in activated CD8 T cells with activation of Th17 T cells, in the context of lower baseline Tregs and greater CLL resistance to duvelisib, is associated with duvelisib-related autoimmune toxicity.

Activation of Notch and Myc Signaling via B-cell-Restricted Depletion of Dnmt3a Generates a Consistent Murine Model of Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is characterized by disordered DNA methylation, suggesting these epigenetic changes might play a critical role in disease onset and progression. The methyltransferase DNMT3A is a key regulator of DNA methylation. Although DNMT3A somatic mutations in CLL are rare, we found that low DNMT3A expression is associated with more aggressive disease. A conditional knockout mouse model showed that homozygous depletion of Dnmt3a from B cells results in the development of CLL with 100% penetrance at a median age of onset of 5.3 months, and heterozygous Dnmt3a depletion yields a disease penetrance of 89% with a median onset at 18.5 months, confirming its role as a haploinsufficient tumor suppressor. B1a cells were confirmed as the cell of origin of disease in this model, and Dnmt3a depletion resulted in focal hypomethylation and activation of Notch and Myc signaling. Amplification of chromosome 15 containing the Myc gene was detected in all CLL mice tested, and infiltration of high-Myc-expressing CLL cells in the spleen was observed. Notably, hyperactivation of Notch and Myc signaling was exclusively observed in the Dnmt3a CLL mice, but not in three other CLL mouse models tested (Sf3b1-Atm, Ikzf3, and MDR), and Dnmt3a-depleted CLL were sensitive to pharmacologic inhibition of Notch signaling in vitro and in vivo. Consistent with these findings, human CLL samples with lower DNMT3A expression were more sensitive to Notch inhibition than those with higher DNMT3A expression. Altogether, these results suggest that Dnmt3a depletion induces CLL that is highly dependent on activation of Notch and Myc signaling. SIGNIFICANCE: Loss of DNMT3A expression is a driving event in CLL and is associated with aggressive disease, activation of Notch and Myc signaling, and enhanced sensitivity to Notch inhibition.

Post-Transformation IGHV-IGHD-IGHJ Mutations in Chronic Lymphocytic Leukemia B Cells: Implications for Mutational Mechanisms and Impact on Clinical Course.

Analyses of IGHV gene mutations in chronic lymphocytic leukemia (CLL) have had a major impact on the prognostication and treatment of this disease. A hallmark of IGHV-mutation status is that it very rarely changes clonally over time. Nevertheless, targeted and deep DNA sequencing of IGHV-IGHD-IGHJ regions has revealed intraclonal heterogeneity. We used a DNA sequencing approach that achieves considerable depth and minimizes artefacts and amplification bias to identify IGHV-IGHD-IGHJ subclones in patients with prolonged temporal follow-up. Our findings extend previous studies, revealing intraclonal IGHV-IGHD-IGHJ diversification in almost all CLL clones. Also, they indicate that some subclones with additional IGHV-IGHD-IGHJ mutations can become a large fraction of the leukemic burden, reaching numerical criteria for monoclonal B-cell lymphocytosis. Notably, the occurrence and complexity of post-transformation IGHV-IGHD-IGHJ heterogeneity and the expansion of diversified subclones are similar among U-CLL and M-CLL patients. The molecular characteristics of the mutations present in the parental, clinically dominant CLL clone (CDC) differed from those developing post-transformation (post-CDC). Post-CDC mutations exhibit significantly lower fractions of mutations bearing signatures of activation induced deaminase (AID) and of error-prone repair by Polη, and most of the mutations were not ascribable to those enzymes. Additionally, post-CDC mutations displayed a lower percentage of nucleotide transitions compared with transversions that was also not like the action of AID. Finally, the post-CDC mutations led to significantly lower ratios of replacement to silent mutations in VH CDRs and higher ratios in VH FRs, distributions different from mutations found in normal B-cell subsets undergoing an AID-mediated process. Based on these findings, we propose that post-transformation mutations in CLL cells either reflect a dysfunctional standard somatic mutational process or point to the action of another mutational process not previously associated with IG V gene loci. If the former option is the case, post-CDC mutations could lead to a lesser dependence on antigen dependent BCR signaling and potentially a greater influence of off-target, non-IG genomic mutations. Alternatively, the latter activity could add a new stimulatory survival/growth advantage mediated by the BCR through structurally altered FRs, such as that occurring by superantigen binding and stimulation.

Multi-Factor Clustering Incorporating Cell Motility Predicts T Cell Expansion Potential.

Expansion of an initial population of T cells is essential for cellular immunotherapy. In Chronic Lymphocytic Leukemia (CLL), expansion is often complicated by lack of T cell proliferation, as these cells frequently show signs of exhaustion. This report seeks to identify specific biomarkers or measures of cell function that capture the proliferative potential of a starting population of cells. Mixed CD4+/CD8+ T cells from healthy donors and individuals previously treated for CLL were characterized on the basis of proliferative potential and in vitro cellular functions. Single-factor analysis found little correlation between the number of populations doublings reached during expansion and either Rai stage (a clinical measure of CLL spread) or PD-1 expression. However, inclusion of in vitro IL-2 secretion and the propensity of cells to align onto micropatterned features of activating proteins as factors identified three distinct groups of donors. Notably, these group assignments provided an elegant separation of donors with regards to proliferative potential. Furthermore, these groups exhibited different motility characteristics, suggesting a mechanism that underlies changes in proliferative potential. This study describes a new set of functional readouts that augment surface marker panels to better predict expansion outcomes and clinical prognosis.

Activation of the MAPK pathway mediates resistance to PI3K inhibitors in chronic lymphocytic leukemia.

Inhibitors of Bruton tyrosine kinase (BTK) and phosphatidylinositol 3-kinase δ (PI3Kδ) that target the B-cell receptor (BCR) signaling pathway have revolutionized the treatment of chronic lymphocytic leukemia (CLL). Mutations associated with resistance to BTK inhibitors have been identified, but limited data are available on mechanisms of resistance to PI3Kδ inhibitors. Here we present findings from longitudinal whole-exome sequencing of cells from patients with multiply relapsed CLL (N = 28) enrolled in trials of PI3K inhibitors. The nonresponder subgroup was characterized by baseline activating mutations in MAP2K1, BRAF, and KRAS genes in 60% of patients. PI3Kδ inhibition failed to inhibit ERK phosphorylation (pERK) in nonresponder CLL cells with and without mutations, whereas treatment with a MEK inhibitor rescued ERK inhibition. Overexpression of MAP2K1 mutants in vitro led to increased basal and inducible pERK and resistance to idelalisib. These data demonstrate that MAPK/ERK activation plays a key role in resistance to PI3Kδ inhibitors in CLL and provide a rationale for therapy with a combination of PI3Kδ and ERK inhibitors.

Preneoplastic Alterations Define CLL DNA Methylome and Persist through Disease Progression and Therapy.

Most human cancers converge to a deregulated methylome with reduced global levels and elevated methylation at select CpG islands. To investigate the emergence and dynamics of the cancer methylome, we characterized genome-wide DNA methylation in pre-neoplastic monoclonal B cell lymphocytosis (MBL) and chronic lymphocytic leukemia (CLL), including serial samples collected across disease course. We detected the aberrant tumor-associated methylation landscape at CLL diagnosis and found no significantly differentially methylated regions in the high-count MBL-to-CLL transition. Patient methylomes showed remarkable stability with natural disease and post-therapy progression. Single CLL cells were consistently aberrantly methylated, indicating a homogeneous transition to the altered epigenetic state, and a distinct expression profile together with MBL cells compared to normal B cells. Our longitudinal analysis reveals the cancer methylome to emerge early, which may provide a platform for subsequent genetically-driven growth dynamics and together with its persistent presence suggests a central role in the normal-to-cancer transition.