Yan, Huihuang

Abstract Multiple myeloma (MM) is the most common blood cancer in African American (AA) individuals. AAs have an increased risk of developing Monoclonal Gammopathy of Undetermined Significance (MGUS) and MM compared to non-Hispanic White (NHW) individuals. While several studies have shown a pivotal role for the immune tumor microenvironment (iTME) in MM pathogenesis, the composition of the iTME in AAs with MM is under investigated. Viably frozen whole bone marrow (BM) aspirates from 128 individuals (53 AA and 75 NHW) with either MM (n=65), MGUS (n=28), smoldering MM (SMM, n=13), or healthy controls (n=22) were evaluated by single cell RNA-sequencing (scRNA-seq) or mass cytometry (CyTOF). We characterized 34 (14 AA and 20 NHW) by scRNA-seq and 94 (39 AA and 55 NHW) by CyTOF. CyTOF samples were stained with Maxpar Direct Immune Profiling Assay Kit (Fluidigm), acquired on a Helios mass cytometer and analyzed in Cytobank. Data were analyzed by logistic equivalent regression model with p<0.05 for significance. ScRNA-seq samples were processed as in Yao, 2022. Data were normalized using scvi-tools and clustered by Uniform manifold approximation and projection (UMAP). Differentially expressed markers between clusters were identified using Model-based Analysis of Single-cell Transcriptomics (MAST) in Seurat R package at FDR < 0.05 and log2 fold change > 0.55. ScRNA-seq of the CD45+ non-tumor lymphocytes revealed 17 immune populations (HSC, erythroblast, MDSC, GMP, CD1c+ DC, CD4+ and CD8+ T cell, CD14+ M1 macrophage, CD16+monocyte, monocyte, pDC, naïve B, pre-B, pro-B, memory B, NK/MAIT). CD3E/D+cells were clustered into naïve/central memory (CCR7+, LEF1+, SELL+), memory (GZMK+), effector (GZMB+, GZMH+, FGFBP2+), g/d (TRDC+), MAIT (SLC4A10+, KLRB1+) and IFN CD8+ T cells (IFIT1+, IFIT2+,IFIT3+). Disease progression was associated with increases in median CD4+ and CD8+ GZMK+ T cells, memory B cells and a reduction in all monocyte clusters. Within the diseased cohort in comparison to NHW patients, AA patients had increased CD3+ T cells (CD4 and CD8+) (AA 57.5% vs. NHW 44.3%) and a reduction in overall monocytes (AA 10.5% vs. 26.5%). These results were validated by CyTOF, which confirmed an increase in CD3+ T cells (AA 53.7% vs. NHW 35.7%, p<0.001) including increases in CD8+ (AA 7.0% vs. NHW 3.2%, p<0.05) and CD4+ terminal effector T cells (AA 5.7% vs. NHW 2.7%, p<0.05) and a reduction in monocytes (AA 18.3% vs. NHW 26.6%) among AAs. After adjusting for age and sex, AAs retained an increase in T cells (OR 1.59 (95%CI 1.2-2.01, p=0.00023)) and a reduction in monocytes (OR 0.88 (95%CI 0.78-1, p=0.045)). Despite the increase in CD8+ T cells in AA patients, we identified reduced GZMK+ T cells and reductions in exhaustion markers (TIGIT, LAG3, TOX) in CD8+ T cells in comparison to NHWs. Nevertheless, the distribution of the immune populations was similar between healthy AA and NHW individuals. Our multiomics approach to characterize the iTME identified significant differences among AA compared to NHW patients with MM. Citation Format: Suganti Shivaram, Hongwei Tang, Huihuang Yan, Shulan Tian, Guyu Qin, Emilie Anderson, Neeraj Sharma, Abiola Bolarinwa, Cinthya Zepeda-Mendoza, Stacey Lehman, Felicia Gomez, Amit Mitra, Surendra Dasari, Taxiarchis Kourelis, Shaji Kumar, Linda Baughn. Characterization of the Immune Tumor Microenvironment in a Diverse Cohort of Patients with Multiple Myeloma [abstract]. In: Proceedings of the Blood Cancer Discovery Symposium; 2024 Mar 4-6; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(2_Suppl):Abstract nr P06.

Abstract High-count monoclonal B-cell lymphocytosis (HCMBL) is a precursor condition to chronic lymphocytic leukemia (CLL). We have shown that among individuals with HCMBL, the CLL-International Prognostic Index (CLL-IPI) is prognostic for time-to-first therapy (TTFT). Little is known about the prognostic impact of somatically mutated genes among individuals with HCMBL. We sequenced DNA from 371 individuals with HCMBL using a targeted sequencing panel of 59 recurrently mutated genes in CLL to identify high-impact mutations. We compared the sequencing results with that of our treatment-naïve CLL cohort (N = 855) and used Cox regression to estimate hazard ratios and 95% confidence intervals (CIs) for associations with TTFT. The frequencies of any mutated genes were lower in HCMBL (52%) than CLL (70%). At 10 years, 37% of individuals with HCMBL with any mutated gene had progressed requiring treatment compared with 10% among individuals with HCMBL with no mutations; this led to 5.4-fold shorter TTFT (95% CI, 2.6-11.0) among HCMBL with any mutated gene vs none, independent of CLL-IPI. When considering individuals with low risk of progression according to CLL-IPI, those with HCMBL with any mutations had 4.3-fold shorter TTFT (95% CI, 1.6-11.8) vs those with none. Finally, when considering both CLL-IPI and any mutated gene status, we observed individuals with HCMBL who were high risk for both prognostic factors had worse prognosis than patients with low-risk CLL (ie, 5-year progression rate of 32% vs 21%, respectively). Among HCMBL, the frequency of somatically mutated genes at diagnosis is lower than that of CLL. Accounting for both the number of mutated genes and CLL-IPI can identify individuals with HCMBL with more aggressive clinical course.

Abstract Monoclonal B-cell lymphocytosis (MBL) progresses to chronic lymphocytic leukemia (CLL) requiring therapy at 1% to 5% per year. Improved prediction of progression would greatly benefit individuals with MBL. Patients with CLL separate into 3 distinct epigenetic subtypes (epitypes) with high prognostic significance, and recently the intermediate epitype has been shown to be enriched for high-risk immunoglobulin lambda variable (IGLV) 3-21 rearrangements, impacting outcomes for these patients. Here, we employed this combined strategy to generate the epigenetic and light chain immunoglobulin (ELCLV3-21) signature to classify 219 individuals with MBL. The ELCLV3-21 high-risk signature distinguished MBL individuals with a high probability of progression (39.9% and 71.1% at 5 and 10 years, respectively). ELCLV3-21 improved the accuracy of predicting time to therapy for individuals with MBL compared with other established prognostic indicators, including the CLL international prognostic index (c-statistic, 0.767 vs 0.668, respectively). Comparing ELCLV3-21 risk groups in MBL vs a cohort of 226 patients with CLL revealed ELCLV3-21 high-risk individuals with MBL had significantly shorter time to therapy (P = .003) and reduced overall survival (P = .03) compared with ELCLV3-21 low-risk individuals with CLL. These results highlight the power of the ELCLV3-21 approach to identify individuals with a higher likelihood of adverse clinical outcome and may provide a more accurate approach to classify individuals with small B-cell clones.

Introduction: Despite the improved clinical outcomes with novel therapies in patients with chronic lymphocytic leukemia (CLL), Richter transformation (RT) of CLL remains a major clinical challenge. Our group showed a selective response (~40%) with the PD-1 blocking antibody pembrolizumab in patients with RT, particularly after prior exposure to ibrutinib (Ding et al, Blood, 2017). It has been established that PD-1 inhibits cytotoxic T-cell functions via the interaction with its ligands, PD-L1 and PD-L2. However, the role of PD-1 in tumor B-cells is understudied. Here we show the expression and functional implications of the PD-1 signaling axis in B-cell pathobiology. Methods: CLL-involved lymph node (LN) (n=26) and RT (with diffuse large B-cell lymphoma [DLBCL] histology) -involved LN (n=20) samples were tested for PD-1 expression by immunohistochemistry (IHC) (mouse clone NAT105, Abcam). Then, PD-1 expression was examined in 10 lymphoma cell lines and 1 CLL-like cell line (MEC-1) by both flow cytometry (FC) and Western blot (WB) analysis. PD-1 overexpression using pLEX-lentiviral packaging system (Thermo Scientific) was evaluated for its impact on in vitro cell cycle regulation and in vivo tumor growth. CRISPR/CAS9 mediated PD-1 knockdown evaluated on cell growth in MEC-1 and Mino cells. Role of PD-1 was further examined on the regulation of cell signaling pathways using human phospho kinase array kit (R&D) and WB analysis. Additionally, gene expression signatures were evaluated by RNA-seq using pLEX-lentiviral-PD-1 transfected DLBCL cell lines OCI-LY7 and OCI-LY19. Results: PD-1 expression was significantly increased in RT-LN compared to CLL-LN (mean ± SEM in RT vs. CLL, 30.6% ± 4.7% vs. 11.5% ± 2.8%, p<0.001) as determined by IHC. PD-1 expression was highest in patients with RT where the last prior CLL therapy was ibrutinib. To test the role of PD-1 in tumor B-cells, its expression was assessed in lymphoma and CLL cell lines. The expression of PD-1 was found to be variable by both FC and WB analysis, but at very low-levels in DLBCL cell lines OCI-LY7 and OCI-LY19. Constitutive lentiviral (pLEX-PD-1)-mediated overexpression of PD-1 in OCI-LY7 and OCI-LY19 cells led to increased cell growth (1.4 and 1.9 fold, respectively, compared to original lines after 4 days of culturing of equal number of cells), which was further confirmed by cell cycle analysis which showed an increase in S phase by 20.4% (p<0.05) and 24.5% (p<0.05) in OCI-LY7 and OCI-LY19 cells, respectively. When the luciferase+ PD-1 overexpressing OCI-LY7 cells (1X10 6) were injected intravenously into NSG mice, increased tumor growth (p<0.01) was observed by bioluminescence imaging after 22 days of follow-up. PD-1 knockdown in MEC-1 and Mino cells (high basal PD-1 expression) led to significant inhibition of cell growth (85% and 79.5%, respectively, p<0.05). Using phospho-kinase array, an overall decrease of phosphorylation was detected on multiple sites of p53 (S392, S15, S46) and Chk-2 (T68) in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. This finding was further confirmed by WB analyses. In addition, decreased total p53 and increased phosphorylation on both SHP-1 and SHP-2 were found in PD-1 overexpressing OCI-LY7 and OCI-LY19 cell lines. SHP-1 (TPI-1) and SHP-1+SHP-2 (TPI-1+TNO155) inhibitor treatment promoted OCI-LY19 cell death and led to recovery of phosphorylation on ATM, Chk-2 and p53 in these cells. RNA sequencing data in PD-1 overexpressing OCI-LY7 and OCI-LY19 cells revealed increased expression of genes that promote cell cycle progression and DNA replication. Conclusion: Our data showed robust PD-1 expression in patients with Richter transformation from CLL to DLBCL. PD-1 overexpression in DLBCL lymphoma cell lines enhanced cell proliferation in vitro and in vivo. Further investigation identified that PD-1 modified the phosphorylation/function of SHP phosphatases and thereby regulated p53 pathways ( Figure 1). These findings indicate that RT with DLBCL cells have the ability to “hijack” the PD-1 pathway which results in downregulation of p53 mediated DNA repair. This novel finding provides for new strategies to further evaluate the interaction of PD-1 signals with DNA repair pathways and suggest potential novel targets for RT.

Abstract Motivation Analyzing the overlap between two sets of genomic intervals is a frequent task in the field of bioinformatics. Typically, this is accomplished by counting the number (or proportion) of overlapped regions, which applies an arbitrary threshold to determine if two genomic intervals are overlapped. By making binary calls but disregarding the magnitude of the overlap, such an approach often leads to biased, non-reproducible, and incomparable results. Results We developed the cobind package, which incorporates six statistical measures: the Jaccard coefficient, Sørensen–Dice coefficient, Szymkiewicz–Simpson coefficient, collocation coefficient, pointwise mutual information (PMI), and normalized PMI. These measures allow for a quantitative assessment of the collocation strength between two sets of genomic intervals. To demonstrate the effectiveness of these methods, we applied them to analyze CTCF’s binding sites identified from ChIP-seq, cancer-specific open-chromatin regions (OCRs) identified from ATAC-seq of 17 cancer types, and oligodendrocytes-specific OCRs identified from scATAC-seq. Our results indicated that these new approaches effectively re-discover CTCF’s cofactors, as well as cancer-specific and oligodendrocytes-specific master regulators implicated in disease and cell type development. Availability and implementation The cobind package is implemented in Python and freely available at https://cobind.readthedocs.io/en/latest/.

Abstract Introduction: The introduction of Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) to target B cell receptor (BCR) pathways in chronic lymphocytic leukemia (CLL) has significantly improved clinical responses. However, the effectiveness of BTKi is limited by the development of adaptive drug resistance. Therefore, there is an urgent need to identify critical biologic features that determine BTKi efficacy in order to develop more effective therapies. Epigenetic aberrations play an important role in tumor initiation, progression, and drug resistance. However, it is currently unknown if BCR signaling is also dependent on epigenetic mechanisms to support oncogenic gene expression and survival in CLL B cells. Methods and Results: To gain new insights into the role of epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in other cohorts at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies show that suppressing BCR signaling by BTKi treatment leads to an alteration of a pro-survival epigenetic signature (defined by histone modifications and chromatin accessibility landscapes) in CLL and that the genes regulated by these chromatin changes are enriched in pathways associated with malignant B cell survival (i.e., BCR signaling and apoptosis pathways). Importantly, disruption of this pro-survival epigenetic signature is necessary for effective BTKi treatment. We further find that the transcription factor (TF) Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) couples the BCR signaling to control this pro-survival epigenetic program that supports CLL B cell survival. In specific, we show that nuclear depletion of NFATc1 is required for BTKi induced epigenetic alteration and effective BTKi treatment. Depletion of NFATc1 disrupts the pro-survival epigenetic signature and inhibits CLL B cells survival, which improves the efficacy of BTKi treatment in CLL. Conclusion: Here using leukemic cells from CLL patients with BTKi treatment, we demonstrate that epigenetic regulators are exploited by the oncogenic signaling pathway to support malignant cell survival. Specifically, NFATc1 is utilized by the BCR signaling pathway to control an epigenetic signature that supports CLL cell survival. This BCR regulated epigenetic machinery can be targeted to maintain and enhance BTKi treatment efficacy in CLL. Citation Format: Zhiquan Wang, Huihuang Yan, Justin C. Boysen, Esteban Braggio, Sameer A. Parikh, Neil E. Kay. The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4725.

Abstract TP53 aberrations, including mutations and deletion of 17p13, are important adverse prognostic markers in chronic lymphocytic leukemia (CLL) but are less studied in high count monoclonal B-cell lymphocytosis (HCMBL), an asymptomatic pre-malignant stage of CLL. Here we estimated the prevalence and impact of TP53 aberrations in 1,230 newly diagnosed treatment-naïve individuals (849 CLL, 381 HCMBL). We defined TP53 state as: wild-type (no TP53 mutations and normal 17p), single-hit (del(17p) or one TP53 mutation), or multi-hit (TP53 mutation and del(17p), TP53 mutation and loss of heterozygosity, or multiple TP53 mutations). Cox regression was used to estimate hazard ratios (HR) and 95% confidence intervals (CI) for time to first treatment and overall survival by TP53 state. We found 64 (7.5%) CLL patients and 17 (4.5%) HCMBL individuals had TP53 mutations with variant allele fraction >10%. Del(17p) was present in 58 (6.8%) of CLL and 11 (2.9%) of HCMBL cases. Most individuals had wild-type (N=1,128, 91.7%) TP53 state, followed by multi-hit (N=55, 4.5%) and then single-hit (N=47, 3.8%) TP53 state. The risk of shorter time to therapy and death increased with the number of TP53 abnormalities. Compared to wild-type patients, multi-hit patients had 3-fold and single-hit patients had 1.5-fold increased risk of requiring therapy. Multi-hit patients also had 2.9-fold increased risk of death compared to wild-type. These results remained stable after accounting for other known poor prognostic factors. Both TP53 mutations and del(17p) may provide important prognostic information for HCMBL and CLL that would be missed if only one were measured.

Abstract Background RBBP4 activates transcription by histone acetylation, but the partner histone acetyltransferases are unknown. Thus, we investigated the hypothesis that RBBP4 interacts with p300 in a complex in glioblastoma (GBM). Methods shRNA silencing of RBBP4 or p300 and RNAseq was used to identify genes co-regulated by RBBP4 and p300 in GBM43 patient-derived xenograft (PDX). RBBP4/p300 complex was demonstrated using proximity ligation assay (PLA) and ChIPseq delineated histone H3 acetylation and RBBP4/p300 complex binding in promoters/enhancers. Temozolomide (TMZ)-induced DNA double strand breaks (DSBs) were evaluated by γ-H2AX and proliferation by CyQuant and live cell monitoring assays. In vivo efficacy was based on survival of mice with orthotopic tumors. Results shRBBP4 and shp300 downregulated 4768 genes among which 1485 (31%) were commonly downregulated by both shRNAs, while upregulated genes were 2484, including 863 (35%) common genes. The pro-survival genes were the top-ranked among the downregulated genes, including C-MYC. RBBP4/p300 complex was demonstrated in the nucleus, and shRBBP4 or shp300 significantly sensitized GBM cells to TMZ compared to the control shNT in vitro (P < .05). Moreover, TMZ significantly prolonged the survival of mice bearing GBM22-shRBBP4 orthotopic tumors compared with control shNT tumors (median shNT survival 52 days vs. median shRBBP4 319 days; P = .001). CREB-binding protein (CBP)/p300 inhibitor CPI-1612 suppressed H3K27Ac and RBBP4/p300 complex target proteins, including C-MYC, and synergistically sensitized TMZ in vitro. Pharmacodynamic evaluation confirmed brain penetration by CPI-1612 supporting further investigation to evaluate efficacy to sensitize TMZ. Conclusions RBBP4/p300 complex is present in GBM cells and is a potential therapeutic target.