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Carlos López-Otín and colleagues report recurrent mutations in POT1 in chronic lymphocytic leukemia. This is the first member of the telomeric shelterin complex reported to be mutated in human cancer. Chronic lymphocytic leukemia (CLL) is the most frequent leukemia in adults 1 , 2 , 3 . We have analyzed exome sequencing data from 127 individuals with CLL and Sanger sequencing data from 214 additional affected individuals, identifying recurrent somatic mutations in POT1 (encoding protection of telomeres 1) in 3.5% of the cases, with the frequency reaching 9% when only individuals without IGHV @ mutations were considered. POT1 encodes a component of the shelterin complex and is the first member of this telomeric structure found to be mutated in human cancer. Somatic mutation of POT1 primarily occurs in gene regions encoding the two oligonucleotide-/oligosaccharide-binding (OB) folds and affects key residues required to bind telomeric DNA. POT1 -mutated CLL cells have numerous telomeric and chromosomal abnormalities that suggest that POT1 mutations favor the acquisition of the malignant features of CLL cells. The identification of POT1 as a new frequently mutated gene in CLL may facilitate novel approaches for the clinical management of this disease.

Targeting Notch signaling has emerged as a promising therapeutic strategy for chronic lymphocytic leukemia (CLL), particularly in NOTCH1-mutated patients. We provide first evidence that the Notch ligand DLL4 is a potent stimulator of Notch signaling in NOTCH1-mutated CLL cells while increases cell proliferation. Importantly, DLL4 is expressed in histiocytes from the lymph node, both in NOTCH1-mutated and -unmutated cases. We also show that the DLL4-induced activation of the Notch signaling pathway can be efficiently blocked with the specific anti-Notch1 antibody OMP-52M51. Accordingly, OMP-52M51 also reverses Notch-induced MYC, CCND1, and NPM1 gene expression as well as cell proliferation in NOTCH1-mutated CLL cells. In addition, DLL4 stimulation triggers the expression of protumor target genes, such as CXCR4, NRARP, and VEGFA, together with an increase in cell migration and angiogenesis. All these events can be antagonized by OMP-52M51. Collectively, our results emphasize the role of DLL4 stimulation in NOTCH1-mutated CLL and confirm the specific therapeutic targeting of Notch1 as a promising approach for this group of poor prognosis CLL patients.

Cyclin D1 is an oncogene frequently overexpressed in human cancers that has a dual function as cell cycle and transcriptional regulator, although the latter is widely unexplored. Here, we investigated the transcriptional role of cyclin D1 in lymphoid tumor cells with cyclin D1 oncogenic overexpression. Cyclin D1 showed widespread binding to the promoters of most actively transcribed genes, and the promoter occupancy positively correlated with the transcriptional output of targeted genes. Despite this association, the overexpression of cyclin D1 in lymphoid cells led to a global transcriptional downmodulation that was proportional to cyclin D1 levels. This cyclin D1-dependent global transcriptional downregulation was associated with a reduced nascent transcription and an accumulation of promoter-proximal paused RNA polymerase II (Pol II) that colocalized with cyclin D1. Concordantly, cyclin D1 overexpression promoted an increase in the Poll II pausing index. This transcriptional impairment seems to be mediated by the interaction of cyclin D1 with the transcription machinery. In addition, cyclin D1 overexpression sensitized cells to transcription inhibitors, revealing a synthetic lethality interaction that was also observed in primary mantle cell lymphoma cases. This finding of global transcriptional dysregulation expands the known functions of oncogenic cyclin D1 and suggests the therapeutic potential of targeting the transcriptional machinery in cyclin D1-overexpressing tumors.

T-cell large granular lymphocytic leukemia (T-LGLL) and chronic lymphoproliferative disorder of natural killer (NK) cells are two infrequent diseases characterized by clonal expansions of cytotoxic T lymphocytes and NK cells, respectively. Somatic mutations of STAT3 are involved in the pathogenesis of these entities. We describe the clinicobiological features, mutational status of STAT3/STAT5B, treatment and outcome of 131 patients. Neutropenia was the most frequent finding at diagnosis, followed by anemia. Concurrent hematological disorders were diagnosed in 37% of patients and autoimmune conditions and solid tumors in 17% and 15%, respectively. All patients who needed treatment belonged to the CD8+CD57+ group. Remarkably, patients included in the CD4+ group had a higher association with solid tumors (p = 0.037). STAT3 mutations were found in 17% of patients, mainly Y640F and D661Y mutations. Patients carrying STAT3 mutations more frequently presented with anemia, neutropenia, high LDH, high large granular lymphocyte counts and need for treatment (p = 0.0037). Methotrexate was the most frequently used agent (72% of cases). The overall response rate to all treatments was 50%. The 10-year overall survival of this series was 78%, with no differences according to the mutational status of STAT3. We compared the survival of these patients with the general Spanish population and no differences were found, confirming the indolent nature of these hematological malignancies. Our study further extends findings documented by others on the clinical behavior of the disease and the impact of STAT3, and for the first time analyzes survival compared to a matched general Spanish population.

INTRODUCTION:Accurate diagnosis of mucinous pancreatic cystic neoplasms (mPCNs) remains a clinical challenge. This study investigated the utility of single GNAS droplet-based digital polymerase chain reaction (ddPCR) analysis as a novel approach to refine the diagnostic accuracy of mPCNs using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA).METHODS:Patients who underwent EUS-FNA and GNAS pancreatic cyst fluid (PCF) analyses for pancreatic cystic lesion (PCL) assessment were prospectively enrolled. Cysts were categorized as mPCNs, non-mPCNs, or inconclusive PCLs (iPCLs) by integrating increasing information levels: high-resolution imaging and non-DNA PCF features (level 1), GNAS PCF analysis (level 2), and surgical pathology (level 3).RESULTS:One hundred forty patients were included, 25 of whom underwent pancreatic surgery. Level 1 identified 68 mPCNs (49%), 24 non-mPCNs (17%), and 48 iPCLs (34%). GNAS mutations were detected in 42 of 68 (62%) mPCNs, 1 of 24 (4%) non-mPCNs, and 16 of 48 (33%) iPCLs. Level 2 increased mPCN detection to 62% and reduced iPCLs by one-third. Mutated GNAS showed 66% sensitivity for diagnosing mPCNs in the whole cohort and 65% in resected cases, outperforming both imaging and non-DNA PCF mucinous criteria, with 100% specificity and limited concordance with carcinoembryonic antigen, cytology, and fluid viscosity, highlighting its complementary diagnostic value. Cost-effectiveness simulations for iPCLs demonstrated that GNAS-ddPCR significantly reduced diagnostic costs by 24% compared with next-generation sequencing testing.DISCUSSION:Single GNAS-ddPCR analysis in PCF supported mPCNs diagnosis in 62% of cases and uncovered 33% of iPCLs as mPCNs with 100% specificity. It adds complementary value to standard cyst fluid markers offering a simple and cost-effective tool for improving PCL diagnosis by EUS-FNA.

The presence of a monoclonal protein detected by serum immunofixation electrophoresis (sIFE) has been reported as an adverse prognostic factor in chronic lymphocytic leukemia (CLL). However, the genetic underpinning of this finding has not been studied. We retrospectively studied 97 CLL patients with simultaneous information on sIFE and genetic alterations detected by next-generation sequencing. sIFE was positive in 49 patients. The most common isotypes were IgG κ (27%) and bi/triclonal (25%). A +sIFE was associated with a higher number of mutated genes [median 2 (range 0–3) vs. 0 (range 0–2), p = 0.006], and a higher frequency of unmutated IGHV status (60 vs. 29%, p = 0.004). An IgM monoclonal protein was associated with TP53 mutations (36% in IgM +sIFE vs. 12% in non-IgM +sIFE or –sIFE, p = 0.04), and bi/triclonal proteins with NOTCH1 mutations (33% in bi/triclonal vs. 9% in monoclonal +sIFE or –sIFE, p = 0.04). These data suggest an association between a +sIFE and a higher mutational burden, and some monoclonal isotypes with specific mutations.

We analyzed 171 patients with asymptomatic IgM monoclonal gammopathies (64 with IgM monoclonal gammopathy of undetermined significance—MGUS and 107 with smoldering Waldenström macroglobulinemia - SWM) who had a bone marrow (BM) evaluation performed at diagnosis. Abnormal free-light chain ratio (53% vs. 31%) and MYD88 mutation prevalence (66% vs. 30%) were higher in patients with SWM. No other differences were found among groups. With a median follow-up of 4.3 years, 14 patients progressed to Waldenström macroglobulinemia, 1 to amyloidosis, and 28 died without progression. The MYD88 mutation was found in 53% of patients (available in 160 patients). Multivariate analysis showed that immunoparesis (subhazard ratio—SHR 10.2, 95% confidence interval—CI: 4.2–24.8; p < 0.001) and BM lymphoplasmacytic infiltration ≥ 20% (SHR: 6, 95% CI: 1.6–22.1; p = 0.007) were associated with higher risk of progression. We developed a risk model based on these two risk factors. In the absence of both variables, an ultra-low risk group was identified (SHR 0.1, 95% CI 0.02–0.5; p = 0.004), with 3% and 6% of cumulative incidence of progression at 10 and 20 years, respectively. Bootstrap analysis confirmed the reproducibility of these results. This study finds immunoparesis and BM infiltration as biomarkers of progression as well as a low-risk group of progression in asymptomatic IgM monoclonal gammopathies.

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and -unmutated cases. In the Eµ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

Chronic lymphocytic leukemia (CLL) is a B lymphoid malignancy highly dependent on the microenvironment. Despite new targeted therapies such as ibrutinib and venetoclax, disease progression and relapse remain an issue. CLL cell interactions with the supportive tissue microenvironment play a critical role in disease pathogenesis. We used a platform for drug discovery based on systems biology and artificial intelligence, to identify drugs targeting key proteins described to have a role in the microenvironment. The selected compounds were screened in CLL cell lines in the presence of stromal cells to mimic the microenvironment and validated the best candidates in primary CLL cells. Our results showed that the commercial drug simvastatin was the most effective and selective out of the tested compounds. Simvastatin decreased CLL cell survival and proliferation as well as cell adhesion. Importantly, this drug enhanced the antitumor effect of venetoclax and ibrutinib. We proposed that systems biology approaches combined with pharmacological screening could help to find new drugs for CLL treatment and to predict new combinations with current therapies. Our results highlight the possibility of repurposing widely used drugs such as statins to target the microenvironment and to improve the efficacy of ibrutinib or venetoclax in CLL cells.