Yan, Huihuang

Abstract Cyclin D1, encoded by CCND1, is a cell cycle regulator, overexpressed in ~15% of multiple myeloma (MM) cases via CCND1::IGH super-enhancer juxtaposition through t(11;14). Cyclin D1 also plays a non-canonical role in transcriptional regulation, but in t(11;14) MM its function in gene regulation remains undefined. We investigated the global transcriptional and epigenomic consequences of CCND1overexpression in t(11;14) MM. To define a CCND1 signature, RNA-seq from two independent primary MM patient cohorts, CoMMpass and Mayo, was utilized to compare high CCND1 t(11;14) cases (≥300 transcripts-per-million [TPM], n=271) vs. low CCND1 hyperdiploid cases (<30 TPM, n=233). Two CCND1 knockout (KO) clones in t(11;14) MM U266B1 were also generated by CRISPR/Cas9, introducing a frameshift insertion (A) after chr11:69641478 (hg38) in exon 1. ATAC-seq and ChIP-seq (H3K4me3, H3K4me1, and H3K27ac) were performed in duplicates, and RNA-seq in triplicates in U266B1 wild-type (WT) and KO. Libraries were sequenced on Illumina NextSeq 2000 (2x51 bp for ChIP/ATAC, 2x150 bp for RNA-seq). Reads were mapped to hg38 by BWA (ChIP/ATAC) or STAR (RNA-seq), peaks called by MACS2 (FDR< 0.01), peak and gene raw counts estimated by featureCounts. Differential peaks (fold-change ≥1.5) and differentially expressed genes (fold-change ≥2) were identified by edgeR (FDR<0.05), including peaks detected in ≥2 samples and genes with TPM ≥0.1 in ≥1 sample. Interactions between promoters and distal regulatory regions were identified using publicly available chromatin interaction data in blood cell types. Pathway analysis was performed using Enrichr. Co-immunoprecipitation was performed in t(11;14) KMS12BM cells. Differential analysis comparing CCND1-high t(11;14) and CCND1-low MM cases in the two patient cohorts identified 21 upregulated and 21 downregulated genes associated with CCND1 expression which were also captured in the wild-typevs. CCND1 KO U266 cells. Enriched pathways (p<0.05) included Notch signaling, early/late estrogen response, epithelial-mesenchymal transition and reduced mitotic spindle and myogenesis. Dysregulated genes included those related to apoptosis, ubiquitin-proteosome, extracellular matrix, adhesion, and immune response, demonstrating a broader role for cyclin D1 in MM beyond cell cycle control. To explore the epigenomic basis of this transcriptional regulation, for each of the 42 differentially expressed genes we examined the changes of histone marks and chromatin accessibility in promoter and the interacting distal regulatory regions in the U266 KO model. Ten of the 21 genes upregulated in CCND1-high (downloaded in KO; e.g., argininosuccinate synthase 1 [ASS1], PMAIP1, MYEOV, CCR7, and DTX1) and 12 of the 21 genes downregulated in CCND1-high (upregulated in KO; e.g., UACA, RASAL2, PTPRZ1, PPT2, and IL23R) showed concordant changes in histone marks and/or chromatin accessibility, predominantly in distal regulatory regions, suggesting a chromatin-mediated mechanism of gene regulation by cyclin D1. The most prominent changes involved H3K27ac, a mark defining active enhancers. As H3K27ac is deposited by histone acetyltransferases CBP, we tested for the possible cyclin D1-CBP interaction. Co-IP confirmed this interaction, suggesting a role for this complex in chromatin remodeling and transcriptional control in t(11;14) MM. One gene notably upregulated in association with CCND1 is ASS1, which encodes a urea cycle enzyme responsible for endogenous arginine synthesis. Metabolomic studies demonstrated KO cells had elevated aspartate and citrulline and lowered arginine and ornithine compared to WT cells (p<0.01) demonstrating impaired urea cycle function. Further, KO cells displayed significantly reduced ASS1 protein expression, cell growth and S-phase entry compared to WT cells in arginine-replete conditions, with both WT and KO cell types further inhibited by arginine depletion (p<0.05). Together, a cyclin D1-associated gene signature was identified supporting its role in t(11;14) MM beyond the CDK4/6 mediated cell cycle control, including a role in metabolic plasticity and survival under arginine depleted conditions. These findings highlight cyclin D1 as a therapeutic vulnerability and open new avenues for personalized treatment strategies.

Background: Multiple myeloma (MM) is an incurable plasma cell malignancy with several recurrent primary cytogenetic abnormalities including t(11,14)(q13;q32), resulting in increased expression of CCND1. Although 40% of patients with t(11;14) MM respond favorably to the BCL-2 inhibitor venetoclax (ven), 60% fail to achieve an objective response. Previous studies have suggested upregulation of PMAIP1, encoding the pro-apoptotic NOXA protein, in association with ven sensitivity (Leblay, Blood, 2024). Here we sought to evaluate the role for NOXA in ven response in t(11;14) MM. Methods: We analyzed whole exome, whole genome and RNA sequencing data of CD138+ tumor cells from 1,359 patients with MM in the CoMMpass and Mayo Clinic cohorts. The histone mark H3K27ac was assessed in t(11;14)+ KMS12PE and U266B1 human myeloma cell lines using ChIP-seq. Gene expression and protein levels of PMAIP1 (NOXA) were also detected by qRT-PCR and Western blot, respectively. Live cell detection was evaluated by Annexin V and PI using flow cytometry and by YoYo3 dye using IncuCyte following treatment with ven. Results: The t(11;14) MM subtype was associated with a 1.6-5.5-fold increased expression of PMAIP1 compared to non-t(11,14) MM in the CoMMpass and Mayo Clinic cohorts (p<0.0001). These findings are consistent with a significant correlation between CCND1 and PMAIP1 expression in both cohorts (p<0.001). To evaluate whether cyclin D1 is directly associated with increasing expression of PMAIP1 in t(11;14) MM, we generated a cyclin D1 knock out (KO) version of the U266B1 line using CRISPR/cas9. Two independent U266B1 KO cell lines showed reduced expression of PMAIP1 and NOXA protein compared to the WT U266B1 consistent with a role for cyclin D1 in regulating PMAIP1 expression. In addition, the KO lines had a 4.8-fold reduced H3K27ac signal at the PMAIP1 locus in comparison to the WT control suggesting a role for cyclin D1 in promoting H3K27ac. We previously observed increased expression of CCND1 in association with the G allele compared to the A allele of rs9344 in both the CoMMpass and Mayo Clinic cohorts (Leukemia, in press). We next asked whether the G risk allele of rs9344 within CCND1 impacted gene expression and H3k27ac levels at the PMAIP1 locus. To study the allele specific effect of CCND1, we used the CRISPR/cas9 edited KMS12PE WT (rs9344 AA) and Knock In (KI) (rs9344 GG) cell lines. In the KI cells, the G allele was accompanied by an ~2-fold increased expression of PMAIP1 and NOXA protein detected by western blot (<0.05). Increased PMAIP1 expression in KI lines was also accompanied by a 2.2-fold increased H3K27ac further suggesting a role for the G allele of rs9344 in promoting H3K27ac and gene expression at the PMAIP1 locus. Finally, we evaluated the consequence of PMAIP1 expression levels in relation to ven response. Increased expression of PMAIP1 and elevated levels of NOXA protein in the KMS12PE KI lines was associated with increased sensitivity to ven resulting in ~50% increase in cell death of KI vs. WT lines (p<0.02). Conclusion: We demonstrate a role for cyclin D1 in regulating PMAIP1 expression through H3K27ac resulting in increased NOXA protein. Levels of NOXA are associated with the G allele of rs9344 and increased venetoclax response.

Introduction: ASXL1-mutant Chronic Myelomonocytic Leukemia (CMML) is a high-risk myeloid neoplasm characterized by epigenetic remodeling and overexpression of leukemogenic driver genes. DLK1 (delta-like homolog 1) is a non-canonical Notch ligand, crucial for the proliferation of early hematopoietic progenitor cells and its expression is controlled by maternal imprinting. DLK1 expression is restricted to select progenitor populations, but was found to be overexpressed in several malignancies including ASXL1-mutant CMML. Here we investigate the molecular mechanisms that drive the aberrant re-expression of DLK1 and postulate potential targeted therapeutic interventions. Methods: We performed bulk RNA- and ChIP-seq (H3K4me1/3, H3K27ac/me3, H2AK119ub, H2BK20ac) on bone marrow mononuclear cells from 20 patients with CMML (11 ASXL1-mutant, 9 -wildtype). All libraries were sequenced on an Illumina NovaSeq instrument. Differential gene expression and histone modification occupancy between ASXL1-mutant and -wildtype patients were assessed. Single-cell RNA-seq was performed for a subset of patients to investigate the cell populations of interest for DLK1 expression. To assess DLK1 allelic expression, we identified a single nucleotide polymorphism (SNP) in the coding region of DLK1 (rs1802710) and used cases which were confirmed heterozygous by Sanger sequencing. DLK1 transcript levels were quantified by RT-PCR, and the relative abundance of alleles was measured using digital droplet PCR. We designed primers to amplify the region around our SNP of interest and utilized high mismatch discrimination locked nucleic acid (LNA) probes labeled with FAM (reference SNP allele) and HEX (alternate SNP allele) to quantify specific alleles. The number of RNA molecules originating from each allele was quantified by counting the number of droplets that fluoresced with the corresponding probe colors. Illumina 850K EPIC microarrays were used to evaluate DNA methylation. Drug studies were carried out in progenitor colony forming assays derived from ASXL1-mutant patients (bone marrow mononuclear cells) with the dual BRD4 and p300 / CBP inhibitor EP31670 (62 nM, 24-hour exposure), quantifying DLK1 expression by real-time RT-PCR. Results: This study included 20 patients with CMML (median age at diagnosis 74 years). All ASXL1 mutations were typical hotspot mutations (most commonly Gly646Trpfs*12) and the variant allele frequencies were compatible with heterozygosity (median 0.41). The spectrum of co-mutations was typical and included TET2 (23%), SRSF2 (16%), CBL (7%), among others. DLK1 was strongly overexpressed in ASXL1-mutant patients (log2 fold-change +4.0, FDR < 0.001). Allele-specific gene expression studies (n = 3) revealed the re-expression of the imprinted maternal DLK1 allele in ASXL1-mutant patients. Single-cell gene expression studies (n = 5) demonstrated that the DLK1 overexpression is restricted to the hematopoietic progenitor cell population. Differential methylation analysis between patients with ASXL1-mutant clonal hematopoiesis (n = 4) and matched controls (n = 8) identified two hypermethylated CpG islands in the DLK1 promoter region (cg18122767, Δß = +8.6%, p = 0.021 and cg15831296, Δß = +5.8%, p = 0.008). Histone modification analysis identified DLK1 as a p300 target gene by mapping H2BK20ac onto established ASXL1-mutant-specific enhancer regions (H3K4me1 + H3K27ac co-occupancy). The distal enhancer EH38D2760182 was predicted to bind p300 and to govern the expression of DLK1. ChIP-seq experiment in K-562 and Kasumi-1 cells showed the binding of BRD4 and p300 to EH38D2760182, respectively. Targeting BRD4 and p300 using EP31670 led to the downregulation of DLK1 expression (mean fold-change = 0.569, p < 0.001, n = 3) in progenitor colony forming assays. Conclusions: We observed the aberrant re-expression of the maternally imprinted DLK1 allele in patients with ASXL1-mutant CMML. Both DLK1 promoter hypermethylation and the genotype-specific activation of enhancers by p300 were identified as putative regulatory mechanisms. The distal enhancer EH38D2760182 was found to bind BRD4 and p300 and the pharmacologic disruption of BRD4 / p300 activity in model systems led to the downregulation of DLK1. The clinical-grade dual inhibitor of BRD and CBP / p300 EP31670 is currently being investigated in patients with ASXL1-mutant chronic myeloid neoplasms (NCT05488548).

Abstract Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples and are hampered by low chromatin yield in FFPE samples due to the lack of reliable and efficient chromatin preparation methods. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. Results: During rehydration of FFPE tissues, applying tissue-level cross-link reversal to the deparaffinized tissue at 65°C dramatically increased chromatin yield in the soluble fraction. We found the resulting chromatin to be compatible with ChIP-qPCR and ChIP-seq analysis of histone marks (H3K4me3, H3K27me3, and H3K27Ac) and RNA polymerase II. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37°C, the majority of soluble chromatin was over 1 kb in size. The soluble chromatin prepared at 45-60°C showed a typical nucleosomal pattern shown in cell lines and frozen tissues. The majority of the chromatin prepared at 65°C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. Conclusions: Our method achieves efficient extraction of high-quality chromatin from FFPE samples suitable for targeted and genome-wide epigenetic assays while minimizing degradation and damages. This approach provides a new platform for developing chromatin-based assays applicable to archived FFPE samples to circumvent the over-fixed nature of FFPE tissues. This work was supported, in part, by National Institutes of Health grant P01DK068055 and the Mayo Clinic Center for Individualized Medicine Epigenomics Program. Citation Format: Jian Zhong, Zhenqing Ye, Chad Clark, Samuel Lenz, Justin Nguyen, Huihuang Yan, Keith Robertson, Gianrico Farrugia, Zhiguo Zhang, Tamas Ordog, Jeong-Heon Lee. Enhanced and controlled chromatin extraction for chromatin-based epigenetic assays in FFPE tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5180.

Abstract MYC dysregulation, the most common genetic aberration in multiple myeloma, is frequently due to the translocation of super-enhancers to the MYC locus. Several drugs target proteins that are enriched at many of these enhancers including BRD4 (BET inhibitors, BETi) and Ikaros (IMiDs), although their mechanism of action remains poorly understood. Here we present a characterization of the responses to these drugs in a collection of over sixty myeloma cell lines having a diversity of MYC rearrangements. We found that the anti-proliferative effects of these drugs significantly correlated with changes in MYC protein levels, consistent with both drugs targeting MYC expression. Despite this common target, there was no statistically significant correlation between the individual BETi and IMiD responses, suggesting that they act through different mechanisms. Of those lines having extremes of sensitivity or resistance, there were two major groups (BETiS/IMiDS and BETiS/IMiDR), a smaller group of four lines resistant to both drugs individually (BETiR/IMiDR) and only one line was BETiR/IMiDS. In the BETiR/IMiDR group, resistance to BETi was mediated by a BRD4-independent mechanism as BRD4 was efficiently released from the MYC-associated enhancers. In all three of BETiR/IMiDR cell lines that we examined, treatment with BETi and IMiD together abolished proliferation and down-regulated MYC, consistent with parallel BRD4- and Ikaros-dependent pathways driving MYC expression. These resistant lines all expressed high levels of the transcription factor ETV4 and knocking out its gene sensitized a line to each drug individually. Thus ETV4 appears to be necessary for the parallel pathways driving MYC expression. There were nine lines in the BETiS/IMiDR group. Sensitivity to BETi in these lines could be explained by either low ETV4 expression or by BETi repressing Ikaros levels (which was only observed in BETiS lines, suggesting that BRD4 drives IKZF1 expression in these lines). Thus, in ETV4-containing lines, BETi sensitivity is due to the simultaneously targeting of the BRD4- and Ikaros-dependent pathways. IMiD resistance likely was due to several reasons. In one cell line, OCIMY5, IMiD had little effect on Ikaros levels, likely due to the previously reported low levels of Cereblon. Seven of the eight remaining lines expressed high levels of either ETV4, or the other potential super-enhancer binding factors IRF4 or RUNX1. In the eight BETiS/IMiDS cell lines, IMiD strongly reduced both Ikaros and Aiolos protein levels, which likely caused IMiD sensitivity. As with the BETiS lines described above, the lines in this group either lacked ETV4 or BETi repressed Ikaros levels. In conclusion, by examining drug response in a collection of genetically annotated myeloma cell lines we have been able to identify factors that contribute the broad range of responses to BETi and IMiDs in myeloma cells. Citation Format: Daniel L. Riggs, Camille Herzog, Victoria M. Garbitt, Niamh Keane, Courtney J. Hillukka, Zachary J. Hammond, Julia E. Wiedmeier, Seth J. Welsh, Shulan Tian, Huihuang Yan, Ranjan Maity, Nizar Bahlis, Paola Neri, W Michael Kuehl, Marta Chesi, P Leif Bergsagel. IMiDs and BET inhibitors target distinct pathways of MYC dysregulation by super-enhancers in multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3015.