Citations

Abstract Background: Lithium (Li) remains the treatment of choice for bipolar disorders (BP). Its mood-stabilizing effects help reduce the long-term burden of mania, depression and suicide risk in patients with BP. It also has been shown to have beneficial effects on disease-associated conditions, including sleep and cardiovascular disorders. However, the individual responses to Li treatment vary within and between diagnostic subtypes of BP (e.g. BP-I and BP-II) according to the clinical presentation. Moreover, long-term Li treatment has been linked to adverse side-effects that are a cause of concern and non-adherence, including the risk of developing chronic medical conditions such as thyroid and renal disease. In recent years, studies by the Consortium on Lithium Genetics (ConLiGen) have uncovered a number of genetic factors that contribute to the variability in Li treatment response in patients with BP. Here, we leveraged the ConLiGen cohort (N=2,064) to investigate the genetic basis of Li effects in BP. For this, we studied how Li response and linked genes associate with the psychiatric symptoms and polygenic load for medical comorbidities, placing particular emphasis on identifying differences between BP-I and BP-II. Results: We found that clinical response to Li treatment, measured with the Alda scale, was associated with a diminished burden of mania, depression, substance and alcohol abuse, psychosis and suicidal ideation in patients with BP-I and, in patients with BP-II, of depression only. Our genetic analyses showed that a stronger clinical response to Li was modestly related to lower polygenic load for diabetes and hypertension in BP-I but not BP-II. Moreover, our results suggested that a number of genes that have been previously linked to Li response variability in BP differentially relate to the psychiatric symptomatology, particularly to the numbers of manic and depressive episodes, and to the polygenic load for comorbid conditions, including diabetes, hypertension and hypothyroidism. Conclusions: Taken together, our findings suggest that the effects of Li on symptomatology and comorbidity in BP are partially modulated by common genetic factors, with differential effects between BP-I and BP-II.

Progression of myeloproliferative neoplasms to blast phase (BPMPN) is associated with lack of response to conventional therapies and dire clinical outcomes. Consequently, there is a major unmet need to develop new therapies for BPMPN. Chromothripsis, the process of catastrophic shattering and haphazard repair of chromosomes, is a key contributor to somatic variation in cancer, but this phenomenon has not yet been described in BPMPN. More broadly, whether chromothripsis might result in actionable molecular events that are amenable to targeting remains an open question. To characterise the contribution of structural variants to BPMPN, we first performed integrated copy number and mutation profiling in 64 BPMPN patients by SNP karyotyping and targeted next generation sequencing. We observed a recurrent pattern of chromothripsis that involved chromosome 21, which together with other structural variants led to amplification of a common region of chromosome 21 (‘chr21amp‘) in ~25% of patients (GISTIC q-val<0.01, Fig 1A). Chr21amp was associated with TP53 mutations and a higher number of copy number alterations. Patients with chr21amp had a particularly aggressive and treatment-resistant phenotype, with 0% surviving 12 months compared to 46% in the non-chr21amp pts ( p=0.0007), retaining significance in multivariate analysis including after correction for TP53 mutation status. Whole genome sequencing confirmed that the chromosomal rearrangements resulting in chr21amp occurred by different mechanisms, ranging from simple amplification to highly complex chromothriptic events involving multiple chromosomes. There were no recurrent translocation partners or mutations. The minimally amplified region (MAR) spanned 2.7Mb and contained 24 genes, with a median copy number of 3.5 (range 2.7-8.3) Single-cell transcriptomics combined with allelic resolution genotyping revealed that chr21amp was present in the dominant subclone and occurred subsequent to JAK2V617F and mut TP53 acquisition. Chr21amp was detectable in phenotypic HSCs and throughout early stages of hematopoiesis, but not in mature erythroid cells, consistent with a differentiation block. Of the 24 genes in the minimally amplified region, only one gene, DYRK1A, a serine threonine kinase linked to cell proliferation and survival, was both differentially expressed (single-cell and bulk RNAseq) and differentially accessible (ATACseq). To explore the functional role of DYRK1A in BPMPN we performed shRNA and CRISPR-mediated DYRK1A-knockdown and knockout (KO) in BPMPN cell lines (HEL and SET-2), which led to impaired cell proliferation. The DYRK1A inhibitors EHT1610 and GNF2133 also led to dose-dependent growth inhibition. DYRK1A-KO BPMPN HEL or SET2 cell clones showed a reduced ability to propagate leukemia in vivo with a significant survival advantage vs. wild type control mice. BPMPN chr21amp+ primary patient CD34+ cells were highly sensitive toDYRK1A inhibitors, while healthy control CD34+ cells were unaffected Prior studies have shown that DYRK1A activates the DREAM complex, a transcriptional repressor of DNA-repair pathways. In chr21amp patient cells, the DREAM DNA repair gene signature was significantly downregulated (NES -1.74, q-val <0.001), while conversely in CRISPR DYRK1A KO SET2 cells the transcriptional DNA repair signature was upregulated (NES 1.76, q-val <0.001). In functional assays, CRISPR DYRK1A KO was protective against DNA damage, with a reduction in γ-H2AX foci after etoposide treatment or irradiation ( p<0.01 for both). A second mechanism of leukemogenesis emerged from geneset enrichment analyses, which suggested enhanced JAK-STAT signaling in chr21amp BPMPN cells and downregulation in the CRISPR DYRK1A KO context. We validated this by showing that DYRK1A overexpression activates and potentiates STAT5B transcriptional activity in a luciferase reporter assay. Finally, we noted that the STAT target BCL2 was selectively upregulated in chr21amp cells. BCL2 inhibition showed strong synergy with DYRK1A inhibitors for induction of BPMPN cell apoptosis (Bliss synergy score 15). Collectively, these findings define the chr21amp event as a novel prognostic biomarker in BPMPN. We pinpoint DYRK1A amplification as a central driver of genomic instability and exacerbated JAK-STAT signalling, for the first time linking chromothripsis to a specific druggable target ( Fig 1B).

Introduction: Chronic myelomonocytic leukemia (CMML) is a myeloid neoplasm with limited therapeutic options and dismal outcomes. Truncating mutations in the epigenetic modifier ASXL1 are common and lead to a proliferative disease phenotype, resistance to epigenetic therapies, and higher rates of AML transformation. ASXL1-mutant CMML is characterized by widespread epigenetic remodeling and up-regulation of key leukemogenic driver genes (e.g. HOXA9 and its co-factor MEIS1). There is a genotype-specific activation of enhancers in ASXL1-mutant CMML that is associated with up-regulation of these leukemogenic driver genes. Here, we test the hypothesis that disrupting the oncogenic interactions at these genotype-specific enhancers can be exploited for therapeutic benefit. Methods: We identified activated enhancers specific to ASXL1-mutant CMML as previously described ( Binder et al. Nat Commun 2022;13(1):1434). To disrupt the E2542605 enhancer, we created unique mapped single guide RNAs (confirmed by BLAT). We generated a ribonucleoprotein complex using the Alt-R CRISPR-Cas9 system (IDT Technologies, Coralville IA). We combined the RNP along with Electroporation Enhancer to 3x10^5 cells of the monocytic cell line U937 in 20uL strip-wells provided by the SF Cell Line 4D-Nucleofector X Kit (Lonza, Basel, Switzerland). To deliver the RNP, U937 cells were electroporated via listed cell line specific protocol by the Amaxa 4D-Nucleofector (Lonza). Forty-eight hours post-electroporation, cells were split and sequenced for E2542605 genomic structure disruption. In parallel, RNA was harvested, converted to cDNA and qPCR for HOXA9 and MEIS1 expression was performed using PrimeTime qPCR FAM labeled probe assays (IDT). Gene expression was determined using delta-delta Ct method. For assessment of selective sensitivity of ASXL1 / enhancer interactions, we performed progenitor colony forming assays using primary bone marrow mononuclear cells from CMML patients with or without ASXL1 mutations and healthy normal controls (n=2 in each category), exposed to increasing concentrations of the dual p300/CBP and BET bromodomain inhibitor EP31670. Briefly, we plated cells in methocellulose fortified with recombinant human cytokines supporting hematopoietic growth in duplicate, in a range of EP31670 concentrations. After 10 days, visible hematopoietic colonies were quantified and characterized. IC 50 values were generated using non-linear regression log(EP31670) vs. normalized response. Results: We computationally identified candidate transcriptional regulators enriched at the activated enhancers specific to ASXL1-mutant CMML, including BRD4 (FDR<2.57x10 -370), CBP (FDR=2.56x10 -217), and p300 (FDR=6.36x10 -93). We selected the ASXL1-mutant-specific enhancer E2542605 (predicted to drive HOXA9 expression), given the up-regulation of HOXA9 in ASXL1-mutant CMML (log 2-fold change +2.05, FDR=0.006, n=14). We disrupted E2542605 using two different CRISPR guides, leading to a PCR trace detection of a 14bp deletion and a 26bp indel in the E2542605 enhancer region, respectively. Both enhancer disruptions led to a down-regulation of its putative target gene HOXA9: Mean relative fold-change 0.36 (p=0.001, n=3) and 0.65 (p=0.046, n=3), respectively ( Fig. 1a). We then used a pharmacologic approach (EP31670) to target the candidate transcriptional regulators involved in the oncogenic interaction at these ASXL1-mutant-specific enhancers. We observed selective sensitivity to growth of CMML colonies (particularly ASXL1-mutant colonies) compared to ASXL1-wild type cases and normal bone marrow controls ( Fig. 1b). Conclusions: In CMML patients, truncating ASXL1 mutations are associated with the activation of genotype-specific enhancers and the recruitment of an extended repertoire of transcriptional regulators including BRD4, CBP, and p300. In a model system, the disruption of an enhancer predicted to govern the expression of the leukemogenic driver HOXA9, led to a significant down-regulation of HOXA9 expression. Pharmacologically disrupting the oncogenic interaction at such enhancers with EP31670 in primary patient samples led to significant therapeutic effects at nanomolar drug concentrations in a genotype-specific manner. Oncogenic interactions at activated enhancers are viable therapeutic targets in ASXL1-mutant CMML and warrant further clinical investigation.

Aim: Patients with R/R LBCL after first-line treatment have poor prognosis. Multiple novel therapies have been approved for R/R LBCL in recent years, but CIT regimens are still commonly used. This study evaluated the effectiveness of CIT and novel therapies in 2L+ in patients with R/R LBCL and the association between patient clinical characteristics and real-world outcomes. Methods: This was a multi-site retrospective observational study of patients aged ≥18 years with R/R LBCL in the Lymphoma Epidemiology of Outcomes (LEO) Consortium of Real-World Evidence (CReWE) cohort (patients followed from 1/1/2015 to 2/15/2023) who were treated with CIT or novel therapy in 2L+. CIT regimens included salvage/palliative chemotherapy, lenalidomide, rituximab, or obinutuzumab, used alone or in combination. Novel therapies included polatuzumab vedotin plus bendamustine and rituximab or variations thereof (i.e., pola-based regimen), tafasitamab plus lenalidomide or variations thereof (tafa-based regimen), and loncastuximab tesirine (lonca). Patients' demographic and clinical characteristics were assessed from the first confirmed R/R disease diagnosis and initiation of 2L+ therapy. When multiple lines of therapy (LOTs) were eligible for inclusion in the analysis, one was randomly selected as the index LOT. Real-world outcomes, determined by investigators at each clinical site, including overall response rate (ORR), complete response (CR) rate, duration of response (DOR), duration of complete response (DOCR), progression-free survival (PFS), and overall survival (OS) were described for all patients in each treatment group and for the subgroup of patients with prior chimeric antigen receptor (CAR) T-cell therapy. Multivariable Cox Proportional Hazards models were used to assess associations between select patient clinical characteristics and real-world outcomes. Results: The study population included patients treated with CIT (N=653), pola-based regimen (N=116), tafa-based regimen (N=55), and lonca (N=42) ( Figure 1A). The median (m) age at date of index treatment initiation across groups ranged from 63 to 74 years. Across groups, most patients had primary refractory disease-i.e., were refractory to the first LOT (69.5% CIT vs. 73.3% pola vs. 58.2% tafa vs. 71.4% lonca), whereas variable proportions had one prior LOT (65.5% vs. 17.2% vs. 43.6% vs. 7.1%) and prior CAR T-cell therapy (5.1% vs. 43.1% vs. 32.7% vs. 47.6%). In the CIT group, ORR was 33.8%, 15.5% of patients achieved CR, mPFS (95% CI) was 1.9 (1.7, 2.1) months, and mOS was 9.1 (8.1, 10.5) months. In the pola group, ORR was 42.2%, 24.1% of patients achieved CR, mPFS was 2.5 (2, 3.2) months and mOS was 7.8 (6.4, 11.8) months. In the tafa group, ORR was 25.5%, 14.5% of patients achieved CR, mPFS was 2.7 (2.1, 4.2) months, and mOS was 8 (5.3, 12.7) months. In the lonca group, ORR was 40.5%, 21.4% of patients achieved CR, mPFS was 3 (2.1, 5.1) months, and mOS was 4.7 (3.7, 10.8) months. Demographics and outcomes for all patients and those who received prior CAR T-cell therapy are summarized in Figure 1A. In the multivariable Cox regression analysis, International Prognostic Index (IPI) risk score ≥3, CIT as the index treatment type, and primary refractory status were significantly associated with time from response to progression/death (P<0.05; Figure 1B). IPI risk score ≥3, CIT as the index treatment type, and primary refractory status were significantly associated with worse PFS. IPI risk ≥3, ≥3 prior LOTs, and primary refractory status were significantly associated with OS. Conclusion: In this large clinical cohort of patients with R/R LBCL with high proportions having primary refractory disease, all common treatments were associated with modest ORR/CR and poor OS. Receiving CIT treatments, higher IPI score, primary refractory status, and higher number of prior LOTs were significantly associated with poor clinical outcomes. A study with larger sample size and longer follow-up may be warranted to further understand the clinical effectiveness of novel treatments. Thus, despite the availability of novel treatments during the study period, these findings highlight the need for more effective treatment alternatives beyond currently available options to improve outcomes.

Background: BMT CTN 1703 was a Phase 3 trial comparing Tac/MTX (n = 217) to post-transplant cyclophosphamide (PT-Cy)/Tac/MMF (n = 214) GVHD prophylaxis after reduced intensity conditioning unrelated donor hematopoietic cell transplant (HCT). PT-Cy significantly improved GVHD-free Relapse-Free Survival (Bolanos-Meade et al NEJM 2023). However overall survival was not significantly different and the # of Grade 2 infections was higher with PT-Cy. To investigate its biologic underpinnings 1703 was linked to a mechanistic study, BMT CTN 1801, which co-enrolled 324 pts (159 CNI/MTX 165 PT-Cy). The resulting dataset enabled an analysis of post-HCT T cell reconstitution at an unprecedented level of detail. Methods: Samples were collected from the HCT infusion and on D+7, 14, 28, 63, 98, 180, 270, 1- and 2-yr. We extracted DNA (2,225 samples) and performed Adaptive Biotech β-strand T Cell Receptor (TCR) Immunosequencing (TCR-Seq). We profiled 44,077 (median) TCRs/sample resulting in 215,155,719 T cells analyzed. We evaluated multiple TCR diversity measures longitudinally and before/after cGVHD and infections. Each measure evaluates a specific aspect of TCR diversity, including Simpsons Clonality (focuses on the most expanded clones) Diversity Slope (mid-range clones) Richness (both mid-range and less-expanded clones) and Singletons (the TCR-Seq equivalent of naïve T cells). This analysis was robust to the full range (1000-50,000) of down-sampled TCRs sequenced. Results: We identified major differences in TCR reconstitution in Tac/MTX vs PT-Cy that begin early and persist through 2 yr post-HCT. These resulted in a significantly less diverse TCR repertoire with PT-Cy with substantial implications for its efficacy and toxicity. The most salient differences are: (1) PT-Cy caused significant in-vivo T cell depletion early post-HCT (% T cells/total WBC at D+28: 8.2% (Tac/MTX) vs 2.2% (PT-Cy) p=2.2x10 -23). (2) The remaining donor T cells in PT-Cy undergo substantial clonal expansion ( Fig 1) resulting in significantly constrained TCR diversity measured by Clonality, Slope, Richness, Singletons (not shown) and Singletons/Richness ( Fig 2) which began early (D+14) and persisted for 2 yr. (3) Linked T cell depletion and clonal expansion resulted in a TCR diversity bottleneck after PT-Cy ( Fig 1). Due to the bottleneck, while the fraction of T cells originating from the transplant infusion at =/> 1 yr was similar between arms (18% vs 24%, p=0.7) there were significantly fewer Singleton TCRs in PT-Cy, suggesting a reduction in naïve T cells ( Fig 2), an outcome that has critical consequences for both cGVHD and infection. Thus while cGVHD onset with Tac/MTX was linked to a significant drop in TCR diversity due to singleton clonal expansion (median log-2 fold change in singletons with Tac/MTX = -0.25 p = 0.01) this drop was not observed with PT-Cy (log-2 fold change = -0.05, p = 0.1). Along with the significantly decreased # singletons at all time-points in PT-Cy ( Fig 2) this suggests that while cGVHD after Tac/MTX is linked to substantial naïve T cell expansion, the same is not true with PT-Cy, identifying a novel mechanism for cGVHD control with PT-Cy. The substantially fewer singletons in PT-Cy vs Tac/MTX may also increase the risk of Grade 2+ infections. T cell responses to new infections rely on antigen recognition/effector maturation of naïve (singleton TCR) T cells. After infection, the TCR repertoire became less diverse for both Tac/MTX and PT-Cy (log-2-fold singleton change -0.17 and -1.2 p = 0.0006 and 0.01) indicating that singletons in both cohorts were capable of expanding to pathogens. However the markedly decreased # singletons in PT-Cy at all timepoints was associated with significantly fewer singletons prior to infection (mean 3979 at 5K read-depth for Tac/MTX vs 2820 for PT-Cy p<0.001). This singleton deficit would place PT-Cy patients at higher risk of missing the critical mass of TCRs required to respond effectively to infections, consistent with their increased rate of Gr 2 infections. Conclusions: We performed longitudinal TCR-Seq on >200 million T cells from 324 HCT patients, representing the largest post-HCT TCR-Seq analysis in the history of the field, and enabling novel insights into TCR dynamics. These data identify a previously unappreciated global TCR diversity bottleneck with PT-Cy that simultaneously protects patients from cGVHD and places them at higher risk of infectious complications.

Background Innovative restructuring of cardiac rehabilitation (CR) delivery remains critical to reduce barriers and improve access to diverse populations. Destination Cardiac Rehab is a novel virtual world technology‐based CR program delivered through the virtual world platform, Second Life, which previously demonstrated high acceptability as an extension of traditional center‐based CR. This study aims to evaluate efficacy and adherence of the virtual world–based CR program compared with center‐based CR within a community‐informed, implementation science framework. Methods Using a noninferiority, hybrid type 1 effectiveness‐implementation, randomized controlled trial, 150 patients with an eligible cardiovascular event will be recruited from 6 geographically diverse CR centers across the United States. Participants will be randomized 1:1 to either the 12‐week Destination Cardiac Rehab or the center‐based CR control groups. The primary efficacy outcome is a composite cardiovascular health score based on the American Heart Association Life's Essential 8 at 3 and 6 months. Adherence outcomes include CR session attendance and participation in exercise sessions. A diverse patient/caregiver/stakeholder advisory board was assembled to guide recruitment, implementation, and dissemination plans and to contextualize study findings. The institutional review board–approved randomized controlled trial will enroll and randomize patients to the intervention (or control group) in 3 consecutive waves/year over 3 years. The results will be published at data collection and analyses completion. Conclusions The Destination Cardiac Rehab randomized controlled trial tests an innovative and potentially scalable model to enhance CR participation and advance health equity. Our findings will inform the use of effective virtual CR programs to expand equitable access to diverse patient populations. Registration URL: https://www.clinicaltrials.gov ; Unique identifier: NCT05897710.

Background: The agrammatic variant of primary progressive aphasia (PAA), primary progressive apraxia of speech (PPAOS), or a combination of both (AOS-PAA) are neurodegenerative disorders characterized by speech-language impairments and together compose the AOS-PAA spectrum disorders. These patients typically have an underlying 4-repeat tauopathy, although they sometimes show evidence of amyloid-β and tau deposition on PET, suggesting Alzheimer’s disease (AD). Given the growing number of pharmacologic treatment options for AD, it is important to better understand the incidence of AD pathology in these patients. Objective: This study aimed to evaluate the frequency of amyloid-β and tau positivity in AOS-PAA spectrum disorders. Sixty-five patients with AOS-PAA underwent a clinical speech-language battery and PiB PET and flortaucipir PET imaging. Methods: Global PiB PET standardized uptake value ratios (SUVRs) and flortaucipir PET SUVRs from the temporal meta region of interest were compared between patient groups. For 19 patients who had died and undergone autopsy, their PET and pathology findings were also compared. Results: The results showed that although roughly half of the patients are positive for at least one biomarker, their clinical symptoms and biomarker status were not related, suggesting that AD is not the primary cause of their neurodegeneration. All but one patient in the autopsy subset had a Braak stage of IV or less, despite four being positive on tau PET imaging. Conclusions: Inclusion criteria for clinical trials should specify clinical presentation or adjust the evaluation of such treatments to be specific to disease diagnosis beyond the presence of certain imaging biomarkers.

Abstract Identification of key phenotypic regions such as necrosis, contrast enhancement, and edema on magnetic resonance imaging (MRI) is important for understanding disease evolution and treatment response in patients with glioma. Manual delineation is time intensive and not feasible for a clinical workflow. Automating phenotypic region segmentation overcomes many issues with manual segmentation, however, current glioma segmentation datasets focus on pre-treatment, diagnostic scans, where treat-ment effects and surgical cavities are not present. Thus, existing automatic segmenta-tion models are not applicable to post-treatment imaging that is used for longitudinal evaluation of care. Here, we present a comparison of three-dimensional convolutional neural networks (nnU-Net architecture) trained on large temporally defined pre-treatment, post-treatment, and mixed cohorts. We used a total of 1563 imaging timepoints from 854 patients curated from 13 different institutions as well as diverse public data sets to understand the capabilities and limitations of automatic segmenta-tion on glioma images with different phenotypic and treatment appearance. We as-sessed the performance of models using Dice coefficients on test cases from each group comparing predictions with manual segmentations generated by trained techni-cians. We demonstrate that training a combined model can be as effective as models trained on just one temporal group. The results highlight the importance of a diverse training set, that includes images from the course of disease and with effects from treatment, in the creation of a model that can accurately segment glioma MRIs at multiple treatment time points.