Biology subjects

Ghobrial, I. M.

Publications and source records attributed to Ghobrial, I. M..

8 recordsLinked to original sources

bioRxiv2023

Systematic characterization of therapeutic vulnerabilities in Multiple Myeloma with Amp1q reveals increased sensitivity to the combination of MCL1 and PI3K inhibitors

The development of targeted therapy for patients with Multiple Myeloma (MM) is hampered by the low frequency of actionable genetic abnormalities. Gain or amplification of chr1q (Amp1q) is the most frequent arm-level copy number gain in patients with MM, and it is associated with higher risk of progression and death despite recent advances in therapeutics. Thus, developing targeted therapy for patients with MM and Amp1q stands to benefit a large portion of patients in need of more effective management. Here, we employed large-scale dependency screens and drug screens to systematically characterize the therapeutic vulnerabilities of MM with Amp1q and showed increased sensitivity to the combination of MCL1 and PI3K inhibitors. Using single-cell RNA sequencing, we compared subclones with and without Amp1q within the same patient tumors and showed that Amp1q is associated with higher levels of MCL1 and the PI3K pathway. Furthermore, by isolating isogenic clones with different copy number for part of the chr1q arm, we showed increased sensitivity to MCL1 and PI3K inhibitors with arm-level gain. Lastly, we demonstrated synergy between MCL1 and PI3K inhibitors and dissected their mechanism of action in MM with Amp1q.

cancer biology↗

bioRxiv2023

Novel mechanism of MYC deregulation in Multiple Myeloma

MYC deregulation occurs in 67% of multiple myeloma (MM) cases and associates with progression and worse prognosis in MM. Enhanced MYC expression is known to be driven by translocation or amplification events, but it only occurs in 40% of MM patients. Here, we describe a new mechanism of MYC regulation, whereby epigenetic regulation of MYC by increased accessibility of a cell-type specific enhancer leads to increased MYC expression. We found enhancer activity does not associate with enhancer hijacking events. We identified specific binding of c-MAF, IRF4, and SPIB transcription factors to the enhancer can activate MYC. In addition, we discovered focal amplification of this specific enhancer in approximately 4% of MM patients. Together, our findings define a new epigenetic mechanism of MYC deregulation in MM beyond known translocations or amplifications and point to the importance of non-coding regulatory elements and their associated transcription factor networks as drivers of MM progression.

cancer biology↗

bioRxiv2023

Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Germinal Center B-cell Diffuse Large B-cell Lymphoma

High expression of MYC and its target genes define a subset of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) associated with poor outcomes. Half of these high-grade cases show chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, while focal deletions of the adjacent non-coding gene PVT1 are enriched in MYC-intact cases. To identify genomic drivers of MYC activation, we used high-throughput CRISPR-interference (CRISPRi) profiling of candidate enhancers in the MYC locus and rearrangement partner loci in GCB-DLBCL cell lines and mantle cell lymphoma (MCL) comparators that lacked common rearrangements between MYC and immunoglobulin (Ig) loci. Rearrangements between MYC and non-Ig loci were associated with unique dependencies on specific enhancer subunits within those partner loci. Notably, fitness dependency on enhancer modules within the BCL6 super-enhancer (BCL6-SE) cluster regulated by a transcription factor complex of MEF2B, POU2F2, and POU2AF1 was higher in cell lines bearing a recurrent MYC::BCL6-SE rearrangement. In contrast, GCB-DLBCL cell lines without MYC rearrangement were highly dependent on a previously uncharacterized 3 enhancer within the MYC locus itself (GCBME-1), that is regulated in part by the same triad of factors. GCBME-1 is evolutionarily conserved and active in normal germinal center B cells in humans and mice, suggesting a key role in normal germinal center B cell biology. Finally, we show that the PVT1 promoter limits MYC activation by either native or heterologous enhancers and demonstrate that this limitation is bypassed by 3 rearrangements that remove PVT1 from its position in cis with the rearranged MYC gene. Key pointsO_LICRISPR-interference screens identify a conserved germinal center B cell MYC enhancer that is essential for GCB-DLBCL lacking MYC rearrangements. C_LIO_LIFunctional profiling of MYC partner loci reveals principles of MYC enhancer-hijacking activation by non-immunoglobulin rearrangements. C_LI

cancer biology↗

bioRxiv2022

Single-cell multi-omics defines the cell-type specific impact of splicing aberrations in human hematopoietic clonal outgrowths

RNA splicing factors are recurrently affected by alteration-of-function mutations in clonal blood disorders, highlighting the importance of splicing regulation in hematopoiesis. However, our understanding of the impact of dysregulated RNA splicing has been hampered by the inability to distinguish mutant and wildtype cells in primary patient samples, the cell-type complexity of the hematopoietic system, and the sparse and biased coverage of splice junctions by short-read sequencing typically used in single-cell RNA sequencing. To overcome these limitations, we developed GoT-Splice by integrating Genotyping of Transcriptomes (GoT) with enhanced efficiency long-read single-cell transcriptome profiling, as well as proteogenomics (with CITE-seq). This allowed for the simultaneous single-cell profiling of gene expression, cell surface protein markers, somatic mutation status, and RNA splicing. We applied GoT-Splice to bone marrow progenitors from patients with myelodysplastic syndrome (MDS) affected by mutations in the most prevalent mutated RNA splicing factor - the core RNA splicing factor SF3B1. High-resolution mapping of SF3B1mut vs. SF3B1wt hematopoietic progenitors revealed a fitness advantage of SF3B1mut cells in the megakaryocytic-erythroid lineage, resulting in an expansion of SF3B1mut erythroid progenitor (EP) cells. SF3B1mut EP cells exhibited upregulation of genes involved in regulation of cell cycle and mRNA translation. Long-read single-cell transcriptomes revealed the previously reported increase of aberrant 3 splicing site usage in SF3B1mut cells. However, the ability to profile splicing within individual cell populations uncovered distinct cryptic 3 splice site usage across different progenitor populations, as well as stage-specific aberrant splicing during erythroid maturation. Lastly, as splice factor mutations occur in clonal hematopoiesis (CH) with increased risk of neoplastic transformation, we applied GoT-Splice to CH samples. These data revealed that the erythroid lineage bias, as well as cell-type specific cryptic 3 splice site usage in SF3B1mut cells, precede overt MDS. Collectively, we present an expanded multi-omics single-cell toolkit to define the cell-type specific impact of somatic mutations on RNA splicing, from the earliest phases of clonal outgrowths to overt neoplasia, directly in human samples.

cancer biology↗

bioRxiv2022

Single-cell multi-omics of human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation

Somatic mutations in cancer genes have been ubiquitously detected in clonal expansions across healthy human tissue, including in clonal hematopoiesis. However, mutated and wildtype cells are morphologically and phenotypically similar, limiting the ability to link genotypes with cellular phenotypes. To overcome this limitation, we leveraged multi-modality single-cell sequencing, capturing the mutation with transcriptomes and methylomes in stem and progenitors from individuals with DNMT3A R882 mutated clonal hematopoiesis. DNMT3A mutations resulted in myeloid over lymphoid bias, and in expansion of immature myeloid progenitors primed toward megakaryocytic-erythroid fate. We observed dysregulated expression of lineage and leukemia stem cell markers. DNMT3A R882 led to preferential hypomethylation of polycomb repressive complex 2 targets and a specific sequence motif. Notably, the hypomethylation motif is enriched in binding motifs of key hematopoietic transcription factors, serving as a potential mechanistic link between DNMT3A R882 mutations and aberrant transcriptional phenotypes. Thus, single-cell multi-omics pave the road to defining the downstream consequences of mutations that drive human clonal mosaicism.

cancer biology↗

bioRxiv2021

Genetic Subtypes of Smoldering Multiple Myeloma are associated with Distinct Pathogenic Phenotypes and Clinical Outcomes

Smoldering multiple myeloma (SMM) is a precursor condition of multiple myeloma (MM) with significant heterogeneity in disease progression. Existing clinical models of progression risk do not fully capture this heterogeneity. Here we integrated 42 genetic alterations from 214 SMM patients using unsupervised binary matrix factorization (BMF) clustering and identified six distinct genetic subtypes. These subtypes were differentially associated with established MM-related RNA signatures, oncogenic and immune transcriptional profiles, and evolving clinical biomarkers. Three subtypes were associated with increased risk of progression to active MM in both the primary and validation cohorts, indicating they can be used to better predict high and low-risk patients within the currently used clinical risk stratification model.

cancer biology↗

bioRxiv2019

In Vivo Monitoring of Rare Circulating Tumor Cell and Cluster Dissemination in a Multiple Myeloma Xenograft Model

We recently developed Diffuse in vivo Flow Cytometry (DiFC), a new pre-clinical research tool for enumerating extremely rare fluorescently-labeled circulating cells directly in vivo. In this paper, we developed a green fluorescent protein (GFP) compatible version of DiFC, and used it to non-invasively monitor the circulating tumor cell (CTC) burden over time in a multiple myeloma disseminated xenograft model. We show that DiFC allowed counting of CTCs at estimated concentrations below 1 cell per mL in peripheral blood with a negligible false alarm rate. DiFC also revealed the presence of CTC clusters in circulation to our knowledge for the first time in this model, and allowed us to calculate their size, kinetics, and frequency of shedding. We anticipate that the unique capabilities of DiFC will have many applications in the study of hematogenous metastasis, and as a powerful complementary methodology to liquid biopsy assays.

bioengineering↗

bioRxiv2018

Inhibition of mitochondrial ferredoxin 1 (FDX1) prevents adaptation to proteotoxic stress

The mechanisms used by cancer cells to resist the severe disruption in protein homeostasis caused by proteasome inhibitors remain obscure. Here, we show this resistance correlates with a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS). Employing small molecule screens, we identified a striking overlap between compounds that preferentially impede the growth of proteasome inhibitor-resistant cancer cells and those that block the growth of high OXPHOS cells. Elesclomol potently exhibits both characteristics. Using genome-wide CRISPR/Cas9-based screening, in vitro validation and NMR spectroscopy we identify mitochondrial protein ferredoxin 1 (FDX1), a critical component of mitochondrial iron-sulfur (Fe-S) cluster biosynthesis, as the primary target of elesclomol. In a mouse model of multiple myeloma, inhibition of FDX1 with elesclomol significantly attenuated the emergence of proteasome inhibitor-resistance and markedly prolonged survival. Our work reveals that the mitochondrial Fe-S cluster pathway is a targetable vulnerability in cancers that are resistant to increased proteotoxic burden.

cancer biology↗