Citations

Abstract Durable T cell immunity against cancer depends on the continual replenishment of effector CD8+ T cells. Thymic output has been correlated with favorable prognosis in cancer patients across a range of ages, suggesting that the thymus is an important source for replenishing T cells capable of controlling cancer progression. However, the effector potential of thymic mature CD8+ T cells and their regulation have not been clearly defined. In this study, we identified the ability of thymic single positive CD8+ T cells to gain effector potential after thymic selection, but they are subject to the regulation of PD-1. We found a previously undisclosed role of PD-1 in limiting both the cytotoxic and exhaustion potential of thymic and peripheral CD8+ T cells. Our results show that although PD-1 inhibition facilitates the expansion of effector CD8+ T cells, effector CD8+ cells gradually lose their antitumor activity within tumor tissues due to advanced exhaustion in the absence of PD-1. Thus, although the preset effector potential in thymic mature CD8+ T cells allows them to rapidly respond to malignant cells in the periphery, PD-1, as a checkpoint, is embedded in the thymic mature CD8+ T cells after positive selection to balance their effector function from exaggeration and exhaustion. Therefore, we propose that a strategy capable of upholding the cytotoxic capacity and avoiding exhaustion of CD8+ T cells during the early stages of PD-1 inhibition therapy is needed to achieve durable antitumor immunity.

AbstractBackgroundIndividuals with spinal cord injury (SCI) commonly have autonomic dysreflexia (AD) with increased sympathetic activity. After SCI, individuals have decreased baroreflex sensitivity and increased vascular responsiveness.ObjectiveTo evaluate the relationship between baroreflex and blood vessel sensitivity with AD symptoms.DesignCase control.SettingTertiary academic center.Patients14 individuals with SCI, 17 matched uninjured controls.InterventionsAll participants quantified AD symptoms using the Autonomic Dysfunction Following SCI (ADFSCI)‐AD survey. Participants received three intravenous phenylephrine boluses, reproducibly increasing systolic blood pressure (SBP) 15–40 mmHg. Continuous heart rate (R‐R interval, ECG), beat‐to‐beat blood pressures (Finapres), and popliteal artery flow velocity were recorded. Vascular responsiveness (α1 adrenoreceptor sensitivity) and heart rate responsiveness to increased SBP (baroreflex sensitivity) were calculated.Main Outcome MeasuresBaroreflex sensitivity after increased SBP; Vascular responsiveness through quantified mean arterial pressure (MAP) 2‐minute area under the curve and change in vascular resistance.ResultsSCI and control cohorts were well matched with mean age 31.9 and 29.6 years (p = .41); 21.4% and 17.6% female, respectively. Baseline MAP (p = .83) and R‐R interval (p = .39) were similar. ADFSCI‐AD scores were higher following SCI (27.9 ± 22.9 vs. 4.2 ± 2.9 in controls, p = .002). To quantify SBP response, MAP area under the curve was normalized to dose/body weight. Individuals with SCI had significantly larger responses (0.26 ± 0.19 mmHg*s/kg*μg) than controls (0.06 ± 0.06 mmHg*s/kg*μg, p = .002). Similarly, leg vascular resistance increased after SCI (24% vs. 6% to a normalized dose, p = .007). Baroreflex sensitivity was significantly lower after SCI (15.0 ± 8.3 vs. 23.7 ± 9.3 ms/mmHg, p = .01). ADFSCI‐AD subscore had no meaningful correlation with vascular responsiveness (R2 = 0.008) or baroreflex sensitivity (R2 = 0.092) after SCI.ConclusionsAlthough this confirms smaller previous studies suggesting increased α1 adrenoreceptor sensitivity and lower baroreflex sensitivity in individuals with SCI, contrary to our hypothesis these differences lacked correlation to increased symptoms of AD. Further research into physiologic mechanisms is needed to explain why some individuals with SCI develop symptoms.

AbstractBackgroundAnti‐amyloid monoclonal antibody therapies for AD have documented plaque removal on amyloid PET scans. We evaluate the findings for amyloid PET, tau PET, FDG PET and volumetric MRI over the course the open label extension (OLE) for the DIAN‐TU gantenerumab treatment, compared to the last imaging obtained prior to the OLE (to evaluate for potential rebound effects) and in comparison to longitudinal imaging in the DIAN Observational study.MethodThis double‐blind, phase 2/3 trial (2012‐2019), followed by open‐label extension (OLE), investigated varying gantenerumab doses up to 1500 mg SQ q2 weeks [NCT NCT01760005]. We report long‐term amyloid‐plaque removal by PET and findings for metabolic FDG PET and tau PET, and additionally changes of atrophy on volumetric MRI. Imaging measures were secondary outcome analyses. Comparison is made between the treatment cohort and controls (DB placebo participants who didn’t enter OLE, plus external controls from the DIAN OBS). Estimated years to onset was included as a covariate. The gap period of ∼1 year is specifically analyzed to estimate any “rebound” effect on amyloid or tau accumulation or neurodegeneration. Volumetric MRI measures include hippocampal volumes, lateral ventricular volumes, and white matter hyperintensities.ResultIn participants who were asymptomatic at baseline and treated with gantenerumab (n = 53), amyloid‐plaque removal measured by PIB‐PET in Centiloids was dose‐dependent. Few participants had reached completely negative levels (below 24). No statistical significance was found for FDG PET, tau PET, or volumetric MRI, including hippocampal volumes. Impact of the gap period of ∼1 year (gap in treatment) on the rate of amyloid accumulation compared to the rate of accumulation predicted in observational studies of this cohort is under evaluation, as are the other imaging measures.ConclusionWhile no overall effect was observed in the total group for non‐amyloid measures, results suggest dose dependent clearance of amyloid plaque in asymptomatic DIAD mutation carriers following long‐term, high‐dose gantenerumab. The potential “rebound effect” of elevated amyloid accumulation for participants for whom there was a significant gap in therapy between the trial and the OLE is under evaluation and will be presented, as will the variables for FDG PET, tau PET, and volumetric MRI.

Abstract Objective To evaluate the effectiveness of multiple decision aid strategies in promoting high quality shared decision making for prevention of stroke in patients with non-valvular atrial fibrillation. Design Cluster randomized controlled trial. Setting Six academic medical centers in the United States. Participants Patient participants were aged ≥18 with a diagnosis of non-valvular atrial fibrillation, at risk for stroke (CHA 2 DS 2 -VASc ≥1 for men, ≥2 for women), and scheduled for a clinical appointment to discuss stroke prevention strategies. Participating clinicians were those who manage stroke prevention strategies for participating patients. Intervention Patients were randomized to use a patient decision aid or usual care; clinicians were randomized to use an encounter decision aid or usual care with all participating patients. Main outcome measures Primary outcome measures were quality of shared decision making measured by OPTION12, knowledge of atrial fibrillation and its management, and decisional conflict. Results 1117 participants across six sites were included in the analysis. Compared with usual care, the combined use of both the patient decision aid and the encounter decision aid improved the quality of shared decision making (adjusted mean difference 12.1 (95% confidence interval (CI) 8.0 to 16.2; P<0.001), improved patients’ knowledge (odds ratio 1.68 (95% CI 1.35 to 2.09; P<0.001), and reduced patients’ decisional conflict (adjusted mean difference −6.3 (95% CI −9.6 to −3.1; P<0.001). Statistically significant improvements were also observed with the encounter decision aid alone versus usual care for all three outcomes and with the patient decision aid alone versus usual care for quality of shared decision making and knowledge. No important differences were observed in treatment choices for stroke prevention or in participants’ satisfaction. No statistically significant difference in the length of visit across study groups was detected. Conclusion Patients who received any decision aid (encounter decision aid, patient decision aid, or both) had lower decisional conflict, better shared decision making, and greater knowledge than those receiving no decision aid, except for the effect of the patient decision aid on decisional conflict, which did not reach statistical significance. The study establishes that use of either pre-visit or in-visit decision aids individually or in combination is advantageous compared with usual care. Trial registration ClinicalTrials.gov NCT04357288 .

Abstract BMT CTN (Blood and Marrow Transplant Clinical Trials Network) 1506 (“MORPHO”) was a randomized study of gilteritinib compared with placebo as maintenance therapy after hematopoietic cell transplantation (HCT) for patients with FLT3-ITD–mutated acute myeloid leukemia (AML). A key secondary end point was to determine the impact on survival of before and/or after HCT measurable residual disease (MRD), as determined using a highly sensitive assay for FLT3-ITD mutations. Generally, gilteritinib maintenance therapy was associated with improved relapse-free survival (RFS) for participants with detectable peri-HCT MRD, whereas no benefit was evident for those lacking detectable MRD. We conducted a post hoc analysis of the data and found that the level of MRD detected with this approach correlated remarkably with RFS and relapse risk, and that MRD detectable at any level negatively affected RFS. In the placebo arm, 42.2% of participants with detectable FLT3-ITD MRD relapsed compared with 13.4% of those without detectable MRD. We found that 14.8% of participants had multiple FLT3-ITD clones detected as MRD and had worse survival irrespective of treatment arm. Finally, we examined the kinetics of FLT3-ITD clonal relapse or eradication and found that participants on the placebo arm with detectable MRD relapsed rapidly after HCT, often within a few weeks. MRD-positive participants on the gilteritinib arm relapsed either with FLT3 wild-type clones (assessed by capillary electrophoresis), after cessation of gilteritinib with persistent MRD, or on progression of multiclonal disease. These data demonstrate the potential of FLT3-ITD MRD to guide therapy with gilteritinib for this subtype of AML. This trial was registered at www.clinicaltrials.gov as #NCT02997202.

ASXL transcriptional regulator 1 (ASXL1) is one of the three most frequently mutated genes in age-related clonal hematopoiesis (CH), alongside DNA methyltransferase 3 alpha (DNMT3A) and Tet methylcytosine dioxygenase 2 ( TET2) . CH can progress to myeloid malignancies including chronic monomyelocytic leukemia (CMML) and is also strongly associated with inflammatory cardiovascular disease and all-cause mortality in humans. DNMT3A and TET2 regulate DNA methylation and demethylation pathways, respectively, and loss-of-function mutations in these genes reduce DNA methylation in heterochromatin, allowing derepression of silenced elements in heterochromatin. In contrast, the mechanisms that connect mutant ASXL1 and CH are not yet fully understood. CH/CMML-associated ASXL1 mutations encode C-terminally truncated proteins that enhance the deubiquitinase activity of the ASXL-BAP1 “PR-DUB” deubiquitinase complex, which removes monoubiquitin from H2AK119Ub. Here, we show that ASXL1 mutant proteins interact with the euchromatic histone lysine methyltransferases 1 and 2 (EHMT1–EHMT2) complex, which generates H3K9me1 and me2, the latter a repressive modification in constitutive heterochromatin. Compared to cells from age-matched wild-type mice, we found that expanded myeloid cells from old (≥18-mo-old) Asxl1tm/+ mice, a heterozygous knock-in mouse model of CH, display genome-wide decreases of H3K9me2, H3K9me3, and H2AK119Ub as well as an associated increase in expression of transposable elements (TEs) and satellite repeats. Increased TE expression was also observed in monocytes from ASXL1 -mutant CMML patients compared to monocytes from healthy controls. Our data suggest that mutant ASXL1 proteins compromise the integrity of both constitutive and facultative heterochromatin in an age-dependent manner by reducing the levels of H3K9me2/3 and H2AK119Ub. This increase in TE expression correlated with increased expression of nearby genes, including many interferon-inducible (inflammation-associated) genes (ISGs).

Introduction: Complex karyotype (CK, ≥3 chromosome (chr) abnormalities (abn)) is an important adverse prognostic indicator in chronic lymphocytic leukemia (CLL). CK at diagnosis (dx) of CLL, particularly with major structural chr abn (i.e. unbalanced translocation, add, marker, ring chr), called type-2 CK (CK2), has been associated with an increased risk of Richter transformation (RT) (Visentin Haematologica 2022). RT is defined as development of aggressive lymphoma, most often large B-cell lymphoma, in the setting of CLL. In patients (pts) with relapsed/refractory CLL treated with ibrutinib, CK as a continuous variable and the presence of polyploidy were independent predictors for RT. CK is a common finding at the time of RT, and high-CK (≥5) has been associated with short survival in RT, although previous studies predominantly included pts with prior chemoimmunotherapy (CIT) administration for their CLL. The prognostic significance of CK in pts with RT whose prior CLL-directed therapies include only non-CIT, as is the current standard of care for CLL, has not been evaluated. Methods: We conducted a multicenter, retrospective analysis of pts from 14 academic centers. Pts with RT diagnosed between 6/2011 to 6/2023, who had CLL-directed treatment that did not include prior CIT, with stimulated karyotype data available, were included. Pts who did not receive treatment for their CLL prior to developing RT were not included. Baseline pt, disease, and cytogenetics data were collected via chart review at time of CLL diagnosis and at the development of RT. Karyotype abn up to 20 were counted on both CLL tissue (peripheral blood or bone marrow) and RT tissue using IWGMC guidelines (Chun Leuk Res 2010). CK was categorized as CK1 (balanced abn, deletion, aneusomies) or CK2. Polyploidy was defined as ≥58 chrs. Karyotype was considered evolved if one or more chr abn were seen in a follow-up sample of CLL tissue that were not seen at CLL dx.TP53 disruption was defined as presence of del(17p) or TP53 mutation. Overall survival (OS) was measured from RT dx and estimated using the Kaplan-Meier method. Cox regression model was used to identify prognostic factors associated with OS. Results: A total of 64 eligible pts were identified. Median age at RT dx was 65 years (range 40-89) and 71.9% of pts were male. Of those evaluated (n=49), 79.6% had unmutated IGVH. Median number of therapies for CLL prior to RT was 1 (range 1-5) and included BTK inhibitor (n=37), BCL2 inhibitor (n=3), both (n=18), or other non-CIT treatment (n=6). At CLL diagnosis, 54.7% of pts had a CK, and the median number of chr abn was 3 (range 0-20). Of those with CK, 96.9% had a CK2, and 6.3% had a polyploid subclone. TP53 disruption was found in 46% of pts. At time of RT, chr analysis of CLL tissue was available for 38 pts and showed 78.9% had a CK, and the median number of chr abn was 8 (range 0-20). Polyploidy was found in 29.6%. TP53 disruption (46 pts evaluated) was found in 52.2%. Karyotype evolution from dx (35 pts evaluated) occurred in 74.3% of pts. In RT tissue, of those evaluated (n=29), 96.6% were clonally related to the preceding CLL. MYC translocation (45 pts evaluated) occurred in 17.8%. CK (20 pts evaluated) was found in 70%, with a median number of chr abn of 8 (range 0-20). Of those with CK, 33.3% had a polyploid subclone. With a median follow-up of 17.8 months (range 2.1-83.0) there were 42 deaths, with a median OS of 14.3 months (range 6.9-29.1). For this subset of patients with karyotype data available who would go on to develop RT, an exploratory univariable analysis for OS from RT found that increasing number of chr abn as a continuous variable at CLL dx was associated with worse OS (HR 1.10, 95% CI 1.02-1.19, p=0.01). Similarly, at RT, increasing number of chr abn in CLL tissue (HR 1.09, 95% CI 1.02-1.16, p=0.01) and in RT tissue (HR 1.09, 95% CI 1.01-1.18, p=0.03) was also prognostic of survival. Karyotype evolution in CLL tissue at RT (HR 3.02, 95% CI 1.02-8.94, p=0.05) was borderline significant for worse OS. Conclusions: Our multi-center study identified increasing number of chr abn at CLL diagnosis and at time of RT, and clonal evolution in the CLL tissue, are associated with worse OS in pts with RT whose CLL was treated without CIT. Limitations include incomplete cytogenetics data across the cohort and potential variability across institutions in cytogenetics laboratory practices. This provides further evidence for the clinical utility of chr analysis in the modern era of CLL therapy.

Introduction: Richter transformation is the development of an aggressive lymphoma, most commonly diffuse large B-cell lymphoma, in patients (pts) with chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Transformation into Hodgkin lymphoma (HT) is a rare event and outcomes in pts with this complication in the current CLL/SLL therapeutic era of small molecule inhibitors (SMI; e.g., Bruton tyrosine kinase inhibitors [BTKi] and BCL2 inhibitors [BCL2i]) are unknown. Methods: We conducted a retrospective study of pts from 12 academic centers. Pts with HT who had not previously received chemotherapy for CLL/SLL-directed treatment (tx) were included. Pt, disease, and tx characteristics were collected. HT was categorized into 3 groups: “Concurrent HT,” defined as HT and CLL/SLL diagnosed simultaneously (within 3 months); “HT without prior CLL/SLL tx,” defined as HT and CLL/SLL diagnosed &gt;3 months apart with the CLL/SLL never treated; and “HT with prior non-chemotherapy tx for CLL/SLL” defined as having received non-chemotherapy CLL/SLL-directed tx prior to the diagnosis (dx) of HT. Overall survival (OS) was measured from HT dx and estimated using the Kaplan-Meier method. Cox regression model was used to identify prognostic factors associated with OS. Results: Within a total cohort of 402 pts with Richter transformation (all histologies) without prior chemotherapy, 41 pts with HT (10%) were identified: 12 with concurrent HT, 10 with HT without prior CLL/SLL tx, and 19 with HT with prior non-chemotherapy tx for CLL/SLL. The median age at CLL/SLL dx was 62 years (range 31-96 years) and 73% were male. At CLL/SLL dx, 67% (14/21 evaluated) had unmutated IGHV, and 24% (8/34 evaluated) had TP53 disruption (del(17p) and/or TP53 mutation). The median age at HT dx was 65 years (range 31-96 years) among all pts. At HT dx, there was no significant difference in highest SUV on PET (median 12.3, range 3-31.1), lactate dehydrogenase (LDH) level (median 242 U/L, range 112-859 U/L), or lymph node longest diameter (median 4.6 cm, range 1.6-25 cm) among the 3 subgroups. The median time from CLL/SLL dx to HT dx was 5.4 years (range 0.3-16.5 years) in pts with HT without prior CLL/SLL tx and 4.2 years (range 0.5-17.3 years) in pts with HT with prior non-chemotherapy tx for CLL/SLL. The median number of prior non-chemotherapy CLL/SLL-directed tx was 1 (range 1-6) and included BTKi only (n=17) and BTKi and BCL2i sequentially (n=2); no patient had received only prior BCL2i. Among the 19 pts with HT with prior non-chemotherapy tx for CLL/SLL, 18 pts were on active tx at time of dx (17 BTKi, 1 BCL2i). Initial HT management included: doxorubicin, bleomycin, vinblastine, dacarbazine (ABVD; n=16), brentuximab vedotin (BV)-AVD (n=10), radiation only (n=2), other systemic tx (n=11), and supportive cares / hospice (n=2). All but 1 of the 11 other systemic tx were various HL tx regimens. Across all pts with available data (n=32), the overall response rate to 1st line HT tx was 78% (69% complete response rate); 13% had progressive disease and 9% (n=3; all with concurrent HT) died within 6 months without formal response assessment. A total of 11 pts received at least 1 additional line of therapy for HT, which included BV (n=4), an immune checkpoint inhibitor (n=1), or both (n=6) at any point beyond 1st line HT tx. Median follow-up from HT dx was 32.8 months (range 2.4-253.8 months). The 36-month OS for the entire cohort was 75% (95% CI: 55-87%). The 36-month OS estimates by subgroups were: 62% (95% CI: 26-84%) in pts with concurrent HT, 100% in pts with HT without prior CLL/SLL tx (no events by 36 months), and 67% (95% CI: 33-87%) in pts with HT with prior non-chemotherapy tx for CLL/SLL. Exploratory univariable analysis identified TP53 disruption at CLL dx (HR 12.36; 95% CI: 2.55-59.84) and lymph node longest diameter (HR 1.32 per 1 cm increase; 95% CI: 1.12-1.55) and LDH 2-fold increase (HR 3.97; 95% CI 1.24-12.68) at HT dx as prognostic for worse OS. HT subgroup was not prognostic. Conclusions: This is the largest report of outcomes in pts with CLL/SLL who develop HT following SMI tx without any prior chemotherapy, representing a contemporary cohort. Prior CLL/SLL tx status was not associated with OS; however, cautious interpretation is required given the sample size. Our findings support the use of appropriate HL-directed treatment in the setting of HT; however, OS estimates remain worse than would be expected in an older adult de novo HL cohort (Cheng Blood Adv 2022).

Introduction The International Prognostic Scoring System (IPSS) and its revised version (IPSS-R) were developed using datasets from patients (pts) with untreated MDS. The cytogenetic risk groups used in IPSS-R were expanded to 5 groups from the 3 used in IPSS. Among the new groups introduced in IPSS-R is the “very complex” karyotype (VCK) where ≥4 karyotypic abnormalities are seen compared to “complex karyotype (CK)” with only 3 abnormalities. However, the prognostic impact of VCK vs CK, as well as individual cytogenetic abnormalities and TP53 mutation (TP53mut) status within the CK and VCK subgroups in pts with MDS treated with hypomethylating agents (HMAs) has not been well studied. Hence, we analyzed these questions in a large cohort of HMA-treated MDS pts in the icMDS VALIDATE database. Methods The icMDS VALIDATE database contains MDS pts treated with frontline HMA monotherapy or HMA-based combinations from 22 specialized international centers. Pts with unknown HMA type, date of HMA initiation, missing age, missing sex, missing cytogenetic data, bone marrow or peripheral blood blasts ≥20%, or missing survival status were excluded. We performed time-to-event analyses using the Kaplan-Meier method and compared groups by the log-rank test. We measured overall survival (OS) from time of HMA initiation. VALIDATE protocol was exempted, acknowledged, or approved by all centers' IRBs. VALIDATE is supported by an independent grant from Abbvie. Results A total of 1940 pts were included. Median age was 70 (range: 18-99) years and 66% were men. According to IPSS-R cytogenetic risk groups, 1.9% (37) were very good, 43.4% (841) good, 15.5% (301) intermediate, 11.6% (226) poor, and 27.6% (535) very poor. By HMA type, 71% of pts were treated with azacitidine, 14% with decitabine, 6% with HMA and venetoclax, and 9% with another HMA combination. Approximately 28% of pts underwent allogeneic hematopoietic stem cell transplant (alloHSCT). The IPSS-R cytogenetic risk groups had different OS (p&lt;0.001)with median OS (mOS; months [mo], 95% CI) as follows: very good (43.4, 34.7-72.5), good (41.7, 36.4-46.8), intermediate (24.5, 23.1-32.4), poor (24.2, 19.2-28.5), and very poor (13.6, 12.5-14.5). The difference remained significant (p=0.001) after censoring at time of alloHSCT with mOS as follows: very good (40.4, 23.2-64.3), good (24.3, 22.2-26.3), intermediate (19.3, 15.1-22.2), poor (12.2, 10.3-14.8), and very poor (9.1, 8.2-19.7). Interestingly, the difference between mOS for intermediate and poor risk cytogenetics became more apparent after censoring at time of alloHSCT. Next, we compared the outcomes of pts with CK and VCK to non-complex karyotype (NCK) MDS. The mOS for pts with VCK MDS (13.6 mo, 12.5-14.5) was worse compared to pts with CK (19.9 mo, 19.9-16.8; p=0.004) and NCK (36.1 mo, 32.7-37.8; p&lt;0.001) MDS. Among the 1458 pts with available molecular data, more pts with VCK (75%, 330) MDS had a concurrent TP53mut compared to pts with CK (40%, 21; p&lt;0.001) and NCK (7%, 64 p&lt;0.001) MDS. Considering only patients with TP53mut MDS, mOS for pts with VCK (12.2, 11.1-13.9) was worse compared with NCK (24.2, 16-1-53.1; p&lt;0.001) but not with CK (19.9, 9.5-NR; p=0.9); the difference between CK and NCK was not significant (p=0.16). Among pts with TP53 wildtype (TP53WT) MDS, VCK (15.0, 13.2-18.2) had a similar mOS to TP53WT CK (18.8, 12.6-41.1; p=0.19) while both groups had worse mOS compared to TP53WT NCK (33.4, 30.1-37.0) (p=0.01). In a survival analysis of 616 pts with MDS with ≥3 cytogenetic abnormalities, -17/del(17p) (12.8 mo; p=0.009), inv(3)/t(3q)/del(3q) (10.9 mo; p=0.006), and del(7q) (12.1mo; p=0.024), though not -7 (12.6mo; p=0.23), were associated with lower mOS compared to the pts with MDS without these abnormalities (14.3mo). Conclusions Using one of the largest databases of HMA-treated MDS pts, we show that IPSS-R cytogenetic risk categories associate with significantly different survivals among HMA-treated MDS pts, even after censoring at time of alloHSCT. Importantly, VCK MDS was associated with worse mOS compared to CK and NCK MDS but not in pts with a concurrent TP53mut. Among pts with MDS with ≥3 chromosomal abnormalities, having -17/del(17p), inv(3)/t(3q)/del(3q), or del(7q) was associated with worse survival.

Oncoprotein-driven human cancers exhibit replication stress (RS), leading to an increase in DNA double-strand breaks (DSB). These DSBs are repaired by one of the three main pathways - homologous recombination repair (HR), non-homologous end-joining (NHEJ), and microhomology-mediated end joining (MMEJ). Cyclin D1 (CCND1) is one of the most overexpressed oncoproteins and is universally overexpressed in mantle cell lymphoma (MCL). While CCND1 overexpression drives cells into the S-phase, its specific effects on RS and DSB repair pathways are poorly understood. Here, we investigated the impact of CCND1 overexpression on RS and DSB repair mechanisms in MCL. First, the expression level of CCND1 positively correlated with heightened RS, as indicated by elevated p-RPA-S33 and p-RPA S8 levels, and increased DNA damage, assessed by g-H2AX, especially in the mitotic phase, across diverse cell models, including MCL. Since CCND1-overexpressed cells proliferate rapidly, we hypothesized that some DNA repair pathways might be upregulated to meet the demand for repairing the excess RS-driven DSBs. Reporter assays demonstrated that the MMEJ pathway was specifically upregulated upon CCND1 overexpression, whereas no significant changes were observed in the HR and NHEJ pathways. Furthermore, CRISPR interference (CRISPRi)-mediated knockdown of CCND1 in MCL cells led to a significant reduction in MMEJ activity. This finding suggests the specificity of CCND1 in mediating the upregulation of the MMEJ pathway. Consistent with this, increased MMEJ activity, assessed via foci formation and immunoblotting of DNA polymerase theta expression (POLQ, encoded by POLQ gene), the primary polymerase for the MMEJ pathway, correlated with CCND1 overexpression, suggesting the crucial role of MMEJ in DSB repair processes under CCND1-overexpressed conditions. Sincethe MMEJ pathwayplays a pivotal role in repairing DSB during mitosis, we assessed the dependency of MCL cells on MMEJ-mediated DSB repair for cell survival. As expected, genetic deletion or pharmacological inhibition (ART558 and novobiocin) of POLQ in MCL cells led to increased RS and increased unrepaired DNA damage during mitosis, ultimately leading to chromosomal instability, mitotic catastrophe, and apoptosis. Additionally, the deficiency of POLQ in MCL cells resulted in increased levels of single-strand DNA, another critical biomarker for RS. Second, we found that ATM deficiency, the second most common genetic alteration after CCND1 overexpression in MCL, further increased the cellular dependence on MMEJ. Interestingly, the accumulation of unrepaired DNA damage during mitosis was notably increased in ATM-deficient CCND1-overexpressed cells. When POLQ was inhibited, a significant antitumor effect was evident in MCL cells, including ibrutinib-resistant MCL, both in vitro and in vivo, and this effect was enhanced by ATM deficiency. Further, simultaneous inhibition of both ATM (AZD0156) and POLQ was synthetic lethal in MCL cell lines and primary tumor cells from patients with MCL. Mechanistically, we observed the direct binding of CCND1 to the POLQ promoter and a corresponding upregulation of POLQ mRNA in CCND1-overexpressing cells. Consistently, a significant overexpression of POLQ mRNA was observed in primary MCL cells, compared to other non-Hodgkin lymphoma cells without CCND1 overexpression. Moreover, the positive correlation between POLQ and CCND1 expression was further validated using syngeneic B-lymphoma cells with and without CCND1 overexpression. Importantly, CRISPRi-mediated CCND1 knockdown reduced POLQ mRNA expression and the dependency of MCL cells on the MMEJ pathway for survival. In addition, ATM deficiency showed a further increase of endogenous POLQ expression and foci formation in cells with CCND1 overexpression, suggesting ATM negatively regulates the MMEJ pathway, especially in the CCND1 overexpressed background. In summary, CCND1 overexpression leads to a substantial increase in RS, intensifying the reliance on MMEJ-mediated mitotic DSB repair. Within MCL, POLQ is crucial in mitigating the adversities of CCND1-driven RS. Our data suggest that targeting the MMEJ pathway via POLQ inhibition, particularly in the presence of CCND1 overexpression and ATM deficiency, emerges as a promising therapeutic approach in oncoprotein-driven hematologic malignancies such as MCL.