Ribosome Biogenesis Research Articles

Abstract A116: TSC1 mutant bladder cancer is characterized by a TSC-associated gene expression signature due to TFE3 transcriptional activity

Abstract Background: Mutations and inactivation of the tumor suppressor gene TSC1 are recurrent (6-10%) events in invasive bladder cancer, but the mechanism by which TSC1 loss promotes bladder cancer development beyond mTORC1 activation is uncertain. The purpose of this study was to assess the functional effects of TSC1 loss in bladder cancer and uncover new therapeutic targets for patients with TSC1 mutations. Materials and methods: We performed differential gene expression and pathway analyses using The Cancer Genome Atlas (TCGA) expression data for TSC1-mutated bladder cancer (TSC1mt-BLCA, n=26) and TSC1 wild-type tumors (TSC1wt-BLCA, n=382) and compared them to an internal cohort of TSC1/TSC2-mutant tumors (n=48). Transcriptomic, epigenetic, and functional analyses, including RNA-seq, H3K27ac ChIP-seq, TFE3 CUT&RUN, ATAC-seq and TFE3 knock-out using CRISPR/Cas9 were conducted on TSC1-mt/wt BLCA cell lines. Results: Comparison of TSC1mt-BLCA and TSC1wt-BLCA TCGA tumors identified 26 genes upregulated in TSC1mt-BLCA out of 60 genes commonly upregulated in Tuberous Sclerosis Complex (TSC) syndromic tumors. Among them, GPNMB showed the greatest increase in expression (median fold change 9.8). GSEA and DESeq2 analyses implicated both mTORC1 hyperactivation and increased lysosomal gene expression in TSC1mt-BLCA. Expression differences in several genes, including GPNMB, and in pS6 were confirmed by immunohistochemistry (IHC) of 5 independent BWH TSC1mt-BLCA and TSC1wt-BLCA specimens each. TFE3, a key regulator of lysosomal gene expression, was elevated in expression and localized to the nucleus in TSC1mt-BLCA by IHC. Molecular studies of TSC1-mt/wt BLCA isogenic cell lines (HCV29, RT4) recapitulated the phenotype found in human tumors. TFE3 was both post-translationally modified (assessed by immunoblot) and predominantly nuclear in TSC1-null cell lines compared to TSC1wt cells, and the localization was partially reversed by rapamycin treatment leading to reduced expression of lysosomal genes. TSC1mt HCV29 cells showed higher expression of multiple genes increased in TSC1mt-BLCA tumors (GPNMB, SQSTM1, GNPDA1). TFE3 knock-out HCV29 clones showed markedly reduced growth, which was not seen with TFE3 KO in TSC1-addback HCV29 cells. GSEA of RNA-seq data indicated that TFE3 KO led to significant reduction in expression of lysosome genes, as well as those involved in ribosome synthesis, DNA replication and DNA excision repair processes. TFE3 CUT&RUN confirmed that TFE3 binds to promoter regions of those genes, thereby directly regulating these pathways. In contrast, TFE3 KO led to an increase in inflammatory and cellular stress genes that could impede TSC1mt cell growth. Conclusions: Our findings show that TSC1mt-BLCA tumors and cell lines have a transcriptional signature similar to that of TSC syndromic tumors and show nuclear localization and transcriptional activation of TFE3. Aberrant TFE3 activation likely contributes to TSC1mt-BLCA development and may therefore be amenable to targeted therapy. Citation Format: Magdalena Losko, Clemens K Probst, Elio Adib, Heng Du, Michelle Hirsch, Krinio Giannikou, David J Kwiatkowski. TSC1 mutant bladder cancer is characterized by a TSC-associated gene expression signature due to TFE3 transcriptional activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A116.

#158 : Whole Transcriptome Analysis of In Vitro-Aged Mouse Oocytes: Effects of Antioxidant Mixture on Oocyte Quality

Background and Aims: Aging is known to have negative impacts on oocyte quality. We reported in the previous meeting that the quality of in vitro-aged oocytes was restored by application of a new antioxidant mixture (AOM). Aiming to understand how AOM influenced on oocyte quality, a whole transcriptome analysis was conducted in this study. Method: Using an in vitro aging model, oocytes were collected from superovulated ICR mice, and part of them were cultured for 5h with (AOM group) or without (Aging group) AOM. Oocytes from fresh oocytes (Control group), Aging group and AOM group were subjected to RNA extraction, amplification, and a genechip microarray analysis. Genes expressed with absolute log2 fold change of [Formula: see text] 2 between two groups were designated as differentially expressed genes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted for further analysis of DEGs. Results: In vitro aging triggered a total of 643 DEGs when compared with the control group. These DEGs were enriched in 19 biological processes, mostly associated with ribosomal synthesis, metabolic process, response to oxidative stress, apoptosis, signal transduction and mRNA processing. These changes are thought to be related to decreasing oocyte quality. AOM effectively prevented age-related change of those DEGs in a broad spectrum. The genes restored by AOM including Rpl7, Rpl35a, Rpl8, Mt1, Fabp2, Acadm, Hadhb, Gstm4, Ncf4, Ppp3ca, Fzd9, Phka2, Spp1 and Lsm6, are highly associated with cellular homeostatic events, suggesting that AOM not only induces changes of response to oxidative stress, but also normalizes gene expression related to cellular activities resulting in improving oocyte quality. Conclusion: This study strongly suggests that AOM restores the gene expression involved in cellular homeostasis. Also, these findings provide new insights of oocyte quality.

Dysregulated bioenergetics pathways in the Alzheimer’s mouse brain

AbstractBackgroundAlzheimer’s disease (AD) is the most common cause of dementia in the aging population. The pathological characteristics include extracellular senile plaques and intracellular neurofibrillary tangles. In addition, mitochondrial dysfunction, oxidative stress, and neuroinflammation contribute to AD pathogenesis.MethodRNA‐seq analysis was done to compare the transcriptome in the brain of 5XFAD mice to the WT brain.ResultThere was significant changes in the expression of genes related to pathways such as, oxidation‐reduction, oxidative phosphorylation, innate immune response, ribosomal protein synthesis, and ubiquitin proteosome system. The most striking feature was the alteration of the oxidation‐reduction process with changes in the expression of a large number of mitochondrial genes. We also observed an upregulation of several immune response genes. Gene interaction network of oxidation‐reduction related genes further confirmed a tight “Gordian knot” of mitochondrial genes. Furthermore, gene interaction analysis of all the 1288 genes showed at least three distinct interaction clusters, with the predominant one relating to cellular energeticsConclusionWe identified 1288 genes distinctly different in the 5XFAD brain compared to the WT brain and found cellular energetics to be the most distinct gene cluster in the AD mouse brain compared to the WT.

Roles of rRNA N-methyladenosine modification in the function of ribosomes.

The N-methyladenosine (m6A) modification of ribosomal RNA (rRNA) plays critical roles in regulating the function of ribosomes, the essential molecular machines that translate genetic information from mRNA into proteins. Specifically, m6A modification affects ribosome biogenesis, stability, and function by regulating the processing and maturation of rRNA, the assembly and composition of ribosomes, and the accuracy and efficiency of translation. Furthermore, m6A modification allows for dynamic regulation of translation in response to environmental and cellular signals. Therefore, a deeper understanding of the mechanisms and functions of m6A modification in rRNA will advance our knowledge of ribosome-mediated gene expression and facilitate the development of new therapeutic strategies for ribosome-related diseases.

Abstract A058: Investigating the molecular role of BRD9 in synovial sarcoma

Abstract Bromodomain-containing protein 9 (BRD9) represents a potential selective vulnerability for tumors with specific alterations in the Brahma-associated factor (BAF) chromatin remodeling complex. One example of such a modification is the incorporation of the SS18-SSX fusion into BAF complexes. This alteration is a hallmark of synovial sarcoma, and thought to drive tumorigenesis. As BRD9 is a subunit unique to ncBAF, tumors that depend on ncBAF for survival may be susceptible to BRD9 degradation. Accordingly, selective BRD9 degradation is a potential therapeutic approach to treat such diseases. Previous work has shown that loss of BRD9 causes cell and tumor proliferation defects (Brien et al. 2018, Michel et al. 2018). To explore the mechanism by which degradation of BRD9 causes cell and tumor proliferation defects in synovial sarcoma, we utilized a multi-omic approach using RNA-seq, ATAC-seq, and ChIP-seq data to understand the molecular impact of loss of BRD9 on relevant cell lines and animal model tumors. ChIP-seq revealed genome-wide colocalization of BRD9 and Myc on chromatin, and BRD9 degradation can lead to MYC loss at the majority of these co-bound regions. Genes commonly regulated in synovial cell lines treated with a BRD9 degrader in vitro and/or in vivo are enriched in MYC target genes, ribosome biogenesis genes and cell cycle genes. Furthermore, the genes commonly downregulated in synovial cell lines treated with a BRD9 degrader have notable MYC binding at their promoter regions. Therefore, we propose that these downregulated genes might be potential MYC targets, specifically in synovial sarcoma, and that downregulation leads to observed proliferation defects. Citation Format: Salih Topal, Claudia Dominici, Michael Collins, David L Lahr, Qianhe Zhou, Flore Uzan, David Mayhew, Ammar Adam, Victoria Garbitt-Amaral, Brandon Antonakos, Oliver Mikse, Hafiz Ahmad, Salonee Parikh, Mei Yun Lin, Hsin-Jung Wu, Gabriel Sandoval, Luis M. M. Soares, Jordana Muwanguzi, Huawei Chen, Zhaoxia Yang, Jason T. Lowe, Matt Netherton, Laura Zawadzke, Johannes Voigt, Liyue Huang, Sabine Ruppel, Ho Man Chan, Ryan Kruger, David S. Millan, Scott Innis, Steven F. Bellon. Investigating the molecular role of BRD9 in synovial sarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A058.

Pan-cancer analysis of 60S Ribosomal Protein L7-Like 1 (RPL7L1) and validation in liver hepatocellular carcinoma

Background and aimsThere is an association between cancer and increased ribosome biogenesis. At present, the RPL7L1 (60S Ribosomal Protein L7-Like 1) were less reported by literature search. Study reports that RPL7L1 is associated with mouse embryonic and skeletal muscle. The study of RPL7L1 on tumors has not been reported. MethodsOur team downloaded the pan-cancer dataset that is uniformly normalized from the UCSC database (N=19131). Our study examined the relationship between RPL7L1 expression level and clinical prognosis with methylation, anti-tumour immunity, functional states, MSI, TMB, DNSss, LOH and chemotherapeutic responses in 43 cancer types and subtypes. Results and conclusionsRPL7L1 was overexpressed in nine tumor types. Gene mutation, tumor microenvironment and methylation modification of RPL7L1 plays a key role in patient prognosis. And the high expression of RPL7L1 was associated with TMB, MSI, LOH especially LIHC and HNSC. We experimentally verified that genes can promote the proliferation and migration of tumor cells. Our study suggested that RPL7L1 biomarker can be used for treating cancer, detecting it, and predicting its prognosis.

Создание генетически кодируемого светоиндуцируемого сенсора для визуализации ядрышка

Nucleolus plays a vital role in enhancing rRNA production and maintaining ribosome biogenesis in tumor cells, making the nucleolus a desirable target for genetic and oncological research. The most convenient method for nucleolus monitoring is fluorescent microscopy, combining high efficiency and accessibility. Nevertheless, currently available fluorescent visualization methods are unsuitable for live-cell monitoring of nucleolus because they require continuous labeling. To address this issue, we have developed a genetically encoded Light-Activated Nucleolus Sensing (LANS) system for real-time nucleolar visualization. The combination of eMags domains and reader domain of DPF3 protein, responsible respectively for the light-induced dimerization and targeting the nucleolus, allowed LANS system to efficiently target nucleolus in several cancer cell lines without affecting cell morphology. This system makes it possible to increase the representation of the LANS2 sample in the nucleolus by 1.5 times relative to the fluorescence intensity values obtained before irradiation of the nucleolus. LANS holds the potential to accelerate the search for new drugs and enhance the primary screening of drug compounds in in vivo models.

Sir2-mediated cytoplasmic deacetylation facilitates pathogenic fungi infection in host plants.

Lysine acetylation is an evolutionarily conserved and widespread post-translational modification implicated in the regulation of multiple metabolic processes, but its function remains largely unknown in plant pathogenic fungi. A comprehensive analysis combined with proteomic, molecular and cellular approaches was presented to explore the roles of cytoplasmic acetylation in Fusarium oxsysporum f.sp. lycopersici (Fol). The divergent cytoplasmic deacetylase FolSir2 was biochemically characterized, which is contributing to fungal virulence. Based on this, a total of 1752 acetylated sites in 897 proteins were identified in Fol via LC-MS/MS analysis. Further analyses of the quantitative acetylome revealed that 115 proteins representing two major pathways, translational and ribosome biogenesis, were hyperacetylated in the ∆Folsir2 strain. We experimentally examined the regulatory roles of FolSir2 on K271 deacetylation of FolGsk3, a serine/tyrosine kinase implicated in a variety of cellular functions, which was found to be crucial for the activation of FolGsk3 and thus modulated Fol pathogenicity. Cytoplasmic deacetylation by FolSir2 homologues has a similar function in Botrytis cinerea and likely other fungal pathogens. These findings reveal a conserved mechanism of silent information regulator 2-mediated cytoplasmic deacetylation that is involved in plant-fungal pathogenicity, providing a candidate target for designing broad-spectrum fungicides to control plant diseases.

Mapping the daily rhythmic transcriptome in the diabetic retina

Retinal function changes dramatically from day to night, yet clinical diagnosis, treatments, and experimental sampling occur during the day. To begin to address this gap in our understanding of disease pathobiology, this study investigates whether diabetes affects the retina's daily rhythm of gene expression. Diabetic, Ins2Akita/J mice, and non-diabetic littermates were kept under a 12 h:12 h light/dark cycle until 4 months of age. mRNA sequencing was conducted in retinas collected every 4 h throughout the 24 hr light/dark cycle. Computational approaches were used to detect rhythmicity, predict acrophase, identify differential rhythmic patterns, analyze phase set enrichment, and predict upstream regulators. The retinal transcriptome exhibited a tightly regulated rhythmic expression with a clear 12-hr transcriptional axis. Day-peaking genes were enriched for DNA repair, RNA splicing, and ribosomal protein synthesis, night-peaking genes for metabolic processes and growth factor signaling. Although the 12-hr transcriptional axis is retained in the diabetic retina, it is phase advanced for some genes. Upstream regulator analysis for the phase-shifted genes identified oxygen-sensing mechanisms and HIF1alpha, but not the circadian clock, which remained in phase with the light/dark cycle. We propose a model in which, early in diabetes, the retina is subjected to an internal desynchrony with the circadian clock and its outputs are still light-entrained whereas metabolic pathways related to neuronal dysfunction and hypoxia are phase advanced. Further studies are now required to evaluate the chronic implications of such desynchronization on the development of diabetic retinopathy.

Proteomics revealed an association between ribosome-associated proteins and amyloid beta deposition in Alzheimer's disease.

Most scholars believe that amyloid-beta (Aβ) has the potential to induce apoptosis, stimulate an inflammatory cascade, promote oxidative stress and exacerbate the pathological progression of Alzheimer's disease (AD). Therefore, it is crucial to investigate the deposition of Aβ in AD. At approximately 6months of age, APP/PS1 double transgenic mice gradually exhibit the development of plaques, as well as spatial and learning impairment. Notably, the hippocampus is specifically affected in the course of AD. Herein, 6-month-old APP/PS1 double transgenic mice were utilized, and the differentially expressed (DE) proteins in the hippocampus were identified and analyzed using 4D label-free quantitative proteomics technology and parallel reaction monitoring (PRM). Compared to wild-type mice, 29 proteins were upregulated and 25 proteins were downregulated in the AD group. Gene Ontology (GO) enrichment analysis of biological processes (BP) indicated that the DE proteins were mainly involved in 'ribosomal large subunit biogenesis'. Molecular function (MF) analysis results were primarily associated with '5.8S rRNA binding' and 'structural constituent of ribosome'. In terms of cellular components (CC), the DE proteins were mainly found in 'polysomal ribosome', 'cytosolic large ribosomal subunit', 'cytosolic ribosome', and 'large ribosomal subunit', among others. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that the results were mainly enriched in the 'Ribosome signaling pathway'. The key target proteins identified were ribosomal protein (Rp)l18, Rpl17, Rpl19, Rpl24, Rpl35, and Rpl6. The PRM verification results were consistent with the findings of the 4D label-free quantitative proteomics analysis. Overall, these findings suggest that Rpl18, Rpl17, Rpl19, Rpl24, Rpl35, and Rpl6 may have potential therapeutic value for the treatment of AD by targeting Aβ.

Translational regulation of cell invasion through extracellular matrix-an emerging role for ribosomes.

Many developmental and physiological processes require cells to invade and migrate through extracellular matrix barriers. This specialized cellular behavior is also misregulated in many diseases, such as immune disorders and cancer. Cell invasive activity is driven by pro-invasive transcriptional networks that activate the expression of genes encoding numerous different proteins that expand and regulate the cytoskeleton, endomembrane system, cell adhesion, signaling pathways, and metabolic networks. While detailed mechanistic studies have uncovered crucial insights into pro-invasive transcriptional networks and the distinct cell biological attributes of invasive cells, less is known about how invasive cells modulate mRNA translation to meet the robust, dynamic, and unique protein production needs of cell invasion. In this review we outline known modes of translation regulation promoting cell invasion and focus on recent studies revealing elegant mechanisms that expand ribosome biogenesis within invasive cells to meet the increased protein production requirements to invade and migrate through extracellular matrix barriers.

The ATF4-RPS19BP1 Axis Modulates Ribosome Biogenesis to Promote Erythropoiesis

The function of hematopoietic stem cells (HSCs) and the process of hematopoiesis are intricately governed by a complex interplay of intrinsic programs and extrinsic signals from the microenvironment (Crane et al., Nat Rev Immunol, 2017). Previous work reported by our group and others has demonstrated that ATF4, a basic region-leucine zipper transcription factor, plays pivotal roles in fetal liver hematopoiesis, HSC maintenance, bone formation and tumorigenesis (Zhao et al., Blood, 2015; Sun et al., Sci Adv, 2021; Yang et al., Cell, 2004). However, the precise function of ATF4 in the bone marrow niche and how ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to conditionally knock out Atf4 in Cdh5 + endothelial cells (ECs), Prx1 + bone marrow stromal cells (BMSCs), Osx + osteo-progenitor cells, and Mx1 + hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. We found that Atf4 deletion in BMSCs reduced BMSC numbers and impaired their CFU-F activity as well as differentiation toward osteoblasts, but spares hematopoiesis and HSC function in adult mice. Furthermore, Atf4 depletion in the ECs or osteo-progenitors of adult mice has a very mild effect on the BM hematopoiesis and HSC maintenance. Intriguingly, Atf4 depletion in the hematopoietic system resulted in severe anemia. More than 70% of the Mx1cre; Atf4 fl/fl (Δ/Δ) mice gradually died from anemia. Atf4 deficiency markedly reduced the frequency of erythroid progenitor cells (EPCs) (including MEP, PreCFU-E, and CFU-E cells) and impaired their CFU-E and BFU-E colony-forming ability. Atf4 depletion severely impaired the repopulation ability of BM cells and the self-renewal capacity of HSCs. Single cell RNA-seq (scRNA-seq) analysis of BM Lin -cKit + cells confirmed the defects of erythropoiesis after Atf4 depletion. The absence of ATF4 compelled early erythroid progenitors to enter the S phase, which led to replication stress and in turn activated both the DNA damage response and apoptosis. Subsequently, we conducted an integrative analysis of the genes that were identified as downregulated in MEPs by RNA-seq, ATAC-seq, and H3K4me3 Cut&Tag and genes that were identified as downregulated in erythroid progenitors (i.e., PreMegE, Ery1, and Ery2) by scRNA-seq, which predicted Rps19bp1 as the top target gene of Atf4. Luciferase reporter and quantitative chromatin immunoprecipitation (qChIP) assays confirmed that ATF4 was a transcriptional activator of Rps19bp1. Downregulation of Rps19bp1 caused by Atf4 deletion led to decreased assembles of 40S proteins, such as RPS3, RPS6 and RPS19. O-propargyl-puromycin (OP-Puro) incorporation and SUnSET assay further confirmed the global protein synthesis defect in Atf4-deficient MEP cells. The protein synthesis defects and impaired erythropoiesis were rescued upon RPS19BP1 overexpression in Atf4 knockdown MEL cell line or BM cKit + cells from Atf4-deficient mice. Ribosome profiling of CMP cells from Δ/Δ and fl/fl mice further demonstrated that the expression of proteins involved in ribosome biogenesis was significantly lower in Atf4-depleted than in wildtype CMP cells. The expression of gene sets linked to PreCFU-E signature was also lower in Atf4-depleted than in wildtype CMPs, suggesting that the reduction in ribosome biogenesis significantly affected the translation efficiency of erythroid-related pathway components, thereby impeding erythroid lineage commitment. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, we demonstrated that, unlike in the fetal liver, ATF4 governs adult HSC function and erythropoiesis in a cell-intrinsic manner. We revealed a novel role for ATF4 in erythropoiesis, which links it to RPS19BP1, ribosome biogenesis and protein translation. Our findings have highlighted the crucial importance of protein synthesis regulation during erythroid lineage commitment. This discovery will likely have extended implications for understanding and treating ribosomopathy-associated erythroid failure.

The RNA Helicase DHX16 Is Required for the Maintenance of Hematopoietic Stem Cells

Lifelong hematopoiesis relies on the homeostasis of hematopoietic stem cells(HSCs), but the underlying mechanisms remain incompletely understood. Here, we identified a critical role for DHX16, a member of DEAH-box RNA helicase, in the maintenance of HSCs. After inducing deletion of Dhx16 gene in mice, the knockout animals developed pancytopenia within 5-7 days and died within two weeks. Flow cytometric analyses demonstrated that Dhx16-deficient mice had significantly reduced hematopoietic stem and progenitor cells, altered cell cycle dynamics with decreased G0 phase and increased G2/M phase, and increased apoptosis of HSCs. Competitive BM transplantation assays convinced the impaired self-renewal ability of Dhx16 knockout mice. To identify Dhx16-dependent genes and molecular pathways involved in the regulation of HSC function, we performed the RNA-Seq and long-read Iso-Seq analysis to compare the transcriptome between the Dhx16-deficient HSCs and Dhx16 fl/fl HSCs. RNA-Seq results showed that signaling pathways in Dhx16-deficient hematopoietic stem cells, including ribosome and spliceosome assembly, G2/M checkpoint, and p53 pathway were significantly affected. While long-read Iso-Seq revealed 210 genes with splicing alterations. Through further study, we confirmed that Dhx16-deficiency led to the retention of intron 4 in Emg1, a gene essential for ribosome biogenesis, resulting in a premature termination and nonsense-mediated mRNA decay. Therewith, decreased Emg1 expression in HSCs impaired ribosome biogenesis, activated the p53 pathway, and resulted in the rapid exhaustion of HSCs. In conclusion, these findings reveal a critical role for DHX16 in HSC survival and function and uncover an unsuspected link between RNA helicases, alternative splicing, ribosome biogenesis, and HSC homeostasis.

A Potential Link between hnRNP K and Cebpα: Implications for Ribosomal Dysfunction in Acute Myeloid Leukemia (AML)

Deletion of the long arm of chromosome 9, del(9q), is a recurrent genetic abnormality involving the haploinsufficient loss of the RNA binding protein hnRNPK and is often associated with poor treatment responses in AML. In addition to reduced hnRNP K expression, other critical proteins, not localized to this locus, are also aberrantly expressed in del9q AML. One protein of critical interest is CEBPα, a well-established myeloid differentiation transcription factor. Recently, CEBPα was discovered to have a novel role in ribosomal RNA (rRNA) synthesis. However, as opposed to CEBPα, hnRNP K's role in RNA stability, rRNA processing, and ribosomal functions is already firmly rooted. Collectively, these results suggest that del(9q) AML dysregulations in hnRNP K and CEBPα expression may have a multi-faceted influence on ribosomal biogenesis and protein translation. Previously, we used animal models and patient samples to demonstrate that Hnrnpk haploinsufficiency alters CEBPa expression, suggesting that reduced hnRNP K expression contributes to decreased CEBPα levels and activity. These findings led us to hypothesize that these distinct, yet interconnected proteins, play a cooperative role in regulating rRNA synthesis in two major ways. Firstly, hnRNP K directly governs rRNA processing and ribosomal loading. And secondly, via hnRNP K's mediated regulation of the nucleolar-localized isoform of CEBPα (p42 isoform), which was recently shown to enhance recruitment of RNA polymerase I to rRNA genes. To explore these relationships, we used AML patient samples, Hnrnpk haploinsufficient animal models, si- and sh- HNRNPK AML cell lines, and biochemical analyses. Initially, RPPA, CyTOF, and western blot analyses were used to investigate how reduced hnRNP K levels affect proteins involved in ribogenesis and protein translation. Here, we observed a direct correlation between hnRNP K expression and defects in proteins and pathways involved in protein translation, such as mTOR, phos-S6, and eEF- and eIF-family members, and CEBPa (log 10pAdj < .0001). Subsequently, we performed polysome assays that revealed 40S and 60S subunit formation was impaired in AML cell lines with reduced hnRNP K levels. Across multiple cell lines, reduced hnRNP K expression directly led to a significant increase in monosomes (unbound/inactive ribosomes; p < 0.01) and a significant decrease in polysome-bound RNA (p < 0.05) . Critically, HNRNPK haploinsufficiency and si- and sh- HNRNPK knock down experiments disrupted the abundance and processing of the 47S transcript (p < 0.05). To understand the impact that hnRNP K has on CEBPα in the context of rRNA biology, we performed co-immunoprecipitation of hnRNP K followed by RNA-seq (RIP-Seq) in human AML cell lines KG-1 and KG-1a. 1076 interacting RNAs were identified, which were strongly enriched in transcription factors implicated in myeloid biology, including CEBPA (log 2pAdj < .0001). Biochemical analyses of sequence-specific motifs conferred direct and functional interactions between hnRNP K and the 5‘ and 3‘ UTRs of the human CEBPA. These interactions support our in vivo findings that del9q AML and Hnrnpk haploinsufficiency lead to reduced CEBPA expression, specifically in the p42 isoform, potentially impacting its role in nucleolar localization and rRNA transcription regulation. Currently, we are evaluating the significance of these interactions at both the biological and translational levels. Using biochemical and cell-based assays, we are evaluating the direct effect that Hnrnpk haploinsufficiency has on rRNA abundance and on the processing of ITS products in the context of CEBPA. Through the use of viability screens, we are examining the efficacy of inhibitors of protein translation, translation initiation, and RNA Pol I on leukemic cells with 9q deletions of reduced hnRNP K expression. Altogether, these findings identify an uncharacterized relationship between del9qAML ( HNRNPK haploinsufficiency), CEBPα expression, and ribogenesis, providing valuable insight towards mechanisms driving this subtype of leukemia. Collectively, these data also suggest that del9q AML represents a novel and currently uncharacterized ribosomopathy.

XPO2 Is Essential for a Subset of AML Cells and Is a Biomarker for Poor Response and Survival in Pediatric and AYA AML

Importins & exportins regulate protein transport across the nuclear membrane. Twenty nuclear transport receptors (NTRs) have been identified; six are involved in nuclear export. NTRs contribute to cancer development, through abnormal localization of protein cargos. The exportin, XPO1, is associated with high risk AML & shorter survival times. However, little is known regarding the functional importance of the related exportin, XPO2 (alias CSE1L). We analyzed a public database of CRISPR/Cas9 depletion screens in 26 AML cell lines. XPO2 ranked in the top 1.4% of essential genes, along with XPO1 (top 1.3%) (DepMap 22Q2 Public+Score, Chronos). An RNAi depletion screen confirmed this, with XPO2 ranked in the top 0.2% of AML genes. Leukemia cells were more dependent on XPO2 vs solid tumors (P = 0.0394) & gene dependency scores indicated a greater reliance on XPO2 vs XPO1 (P <0.0001). Increased expression of XPO2 mRNA was associated with decreased event free survival (EFS) (P = 0.0014) & overall survival (OS) (P = 0.00091) in pediatric & adolescent/young adult (AYA) AML patients (TARGET dataset). In the BeatAML dataset, increased XPO2 mRNA expression was associated with decreased OS in patients <40 years, but not in patients >40 years (P <0.0001). We analyzed protein expression databases & identified XPO2 as the highest expressed exportin in AML cells lines & patient samples. Increased XPO2 expression was detected in 7/7 primary AML samples & 9 AML cell lines by immunoblot (HL60, MV4-11, MOML13, NB4, OCI-AML2, OCI-M2, TEX, U937 & 1254 cells), compared to normal hematopoietic cells. Expression of XPO2 was increased in younger (<40 years) vs older patients (>70 years) (P = 0.0454). Genetic silencing of XPO2 decreased growth & viability of OCI-AML2, TEX, NB4 & 1254 cells. Gambogic acid is an inhibitor of XPO2-mediated nuclear transport. In OCI-AML2, TEX & NB4 cell lines, gambogic acid decreased viability of AML cells (TEX IC50 = 369nM, OCI-AML2 IC50 = 433nM, NB4 IC50 = 161nM). Next, we interrogated a BioID database (PXD007976) & performed gene set enrichment analysis on XPO2 targets. Processes related to ribosome biogenesis were enriched in XPO2 cargo, whereas XPO1 cargo were enriched for snRNA transcription processes (P <0.05). A gene expression signature encompassing XPO2 & its associated cargo genes was enriched in functionally defined LSCs (P = 0.001, in patients with more primitive AML subtypes (P < 2.22 x 10 -16), in more primitive AML blasts as defined by FAB subtype and in high risk subsets, including WT1 mutated pediatric AML. Additionally, the XPO2 gene signature was associated with minimal-residual-disease (MRD) positive disease after induction cycle I (P < 5.1x 10 -6) and II (P = 0.0013), decreased complete remission (CR) rates after cycle I (P = 0.00025) and II (P = 0.02), worse EFS (P = 0.019) and worse OS (P = 0.02) in pediatric and AYA AML patients. In summary, we identified XPO2 as an essential gene for AML . XPO2 is increased in AML cells & primary samples from younger patients. Increased XPO2 expression & increased expression of XPO2 mediated processesare associated with worse outcomes in pediatric AML & correlate with LSC enrichment & primitive AML subtypes. XPO2 may function to enhance ribosomal biogenesis, demonstrating a critical role in leukemogenesis. Thus, this work demonstrates a new dependency of pediatric AML on XPO2 highlighting a new target and biomarker for this disease.

Acute Myeloid and Lymphoblastic Leukemias: A NPM1 Targeting Strategy

Leukemia, a wide spectrum of diseases with altered proliferation and differentiation capacity of myeloid and lymphoid blood progenitors, is the most frequent type of cancer in children and one of the most common in adults. We discovered a covalent small-molecule “probe” for the treatment of acute myeloid and lymphoblastic leukemias that allows for post-translational modulation of the proteome in these leukemia cells. The probe dramatically induces apoptosis of leukemic cells at sub-toxic doses and consistently reduces proliferation, impairs cellular metabolism and promotes chemosensitization to “standard-of-care” chemotherapy regimens. Our proteomic analysis found specific targets that are critical to the leukemia cell survival, namely Nucleophosmin (NPM1) protein, which is a nucleolar phosphoprotein that performs diverse biological functions including molecular chaperoning, ribosome biogenesis, DNA repair, and genome stability. NPM1 gene mutations represent the most common genetic lesion in adult acute myeloid leukemia and their prognostic relevance earned them recognition as a distinct entity in the 2017 World Health Organization (WHO) classification of hematopoietic neoplasms. Our findings suggest a critical role of covalent inhibition of NPM1 in maintaining acute myeloid and lymphoblastic leukemia cells survival. This finding provides a rationale for therapeutic NPM1 modulation in acute leukemias.

Stem-Cell Enriched Cellular Hierarchy of TP53 Mutant Acute Myeloid Leukemia Is Vulnerable to Targeted Protein Degradation of c-MYC

Deregulation of MYC genes occurs in up to 70% of all human cancers and is associated with hallmarks of cancer including mitochondrial and ribosomal biogenesis, cell cycle progression, and metabolic abnormalities. TP53 regulates MYC while MYC suppresses TP53, suggesting counteracting negative feedback loops. Therefore, MYC or its function can be activated when TP53 is not functional. TP53 mutations occur in 30% of relapsed/refractory acute myeloid leukemias (AMLs) patients' survival is dismal, and there are no effective therapies for these patients. Compared to TP53 wild-type (TP53wt), TP53 mutant (TP53mut) AMLs have lower percentages and numbers of leukemia blasts with increased immature CD34+ cells and resistance to chemo- or molecularly targeted therapies. However, the exact cellular hierarchy of TP53mut AML has not been elucidated. We observed significantly increased MYC mRNA levels in (TP53mut), as compared to TP53wt AML, and also increased levels in TP53mut versus TP53wt AML leukemia stem cell (LSC) fractions. This finding was confirmed in a dataset from the Munich Leukemia Laboratory (N = 732). We found significantly upregulated MYC pathways in TP53mut compared to TP53wt AML LSC. We confirmed the increased MYC mRNA levels at the protein level in TP53mut AML by single-cell mass cytometry (CyTOF). To dissect the cellular hierarchy in TP53mut AML, we performed single-cell RNA sequencing of 32 BM samples from healthy donors (N = 3), newly diagnosed, high-risk TP53wt (N = 7) and TP53mut (N = 22) AML patients, with 6,685, 10,687 and 10,687 cells from normal, TP53wt and TP53mut AML bone marrow (BM) cells, respectively. We found highly enriched HSC-like cells and reduced progenitor- and GMP-like cells in TP53mut AML compared to normal BM (NBM) and TP53wt AML samples. We overlayed MYC expression levels on the mapping of cellular components and found higher MYC levels in HSC-like cells in TP53mut AML compared to HSC and HSC-like cells in NBM and TP53wt AML samples, suggesting enrichment of immature HSC-like cells and increased activity of MYC in TP53mut AML LSCs ( Fig. A). To target c-MYC and MYC signaling, we utilized GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC protein (Nishida, ASH 2022). CyTOF confirmed the presence of much increased c-MYC protein levels in primary CD34+ AML than in CD34+ NBM hematopoietic stem cells (HSCs). Notably, CD34+ AML cells showed much greater sensitivity to GT19715 compared to CD34+ NBM cells. Data suggest on-target activity of GT19715 against c-MYC in LSCs with a therapeutic window between LSCs vs. NBM HSCs. Intriguingly, c-MYC protein levels are higher in CD34+CD38+ than in CD34+CD38- AML cells, suggesting that c-MYC drives the proliferation of AML progenitor cells differentiating from quiescent LSCs. Consequently, CD34+CD38+ AML progenitor cells exhibited greater sensitivity to GT19715 compared to CD34+CD38- LSCs. Using paired, TP53 null HL-60 GT19715-sensitive and -resistant cells generated through chronic exposure to GT19715, we interrogated the impact of GT19715 on metabolic changes. GT19715 induced pronounced reductions in basal and maximal oxygen consumption rates (OCRs) in GT19715-sensitive HL-60 cells. GT19715 reduced both basal, glutamine- and glucose-dependent extracellular acidification rates (ECARs) in GT19715-sensitive cells while no inhibition in OCRs or ECARs was observed in GT19715-resistant cells. GT19715 severely inhibited ECARs even after adding glucose to GT19715-sensitive cells, suggesting irreversible inhibition of glycolysis as one of the mechanisms of action of GT19715. GT19715 profoundly reduced AML blasts in TP53mut AML samples (N = 3) ( Fig. B), and in a very aggressive patient-derived xenograft (PDX) TP53mut AML model established from a patient with TP53 p.Y220C and p.P151A mutations along with MECOM rearrangement and K/NRAS mutations. In humanized Crbn I391V mice, where the Crbn-mediated protein degradation is operational, GT19715 only reduced WBC counts along with minimal body weight loss. GT19715 but did not reduce total mouse BM CD45+ cells, suggesting favorable toxicity profiles of GT19715. In conclusion, TP53mut AML comprised highly enriched LSC populations compared to TP53wt AML and targeting of c-MYC protein is highly effective in TP53mut AML in vitro and in vivo with a therapeutic window between AML LSC and normal hematopoietic cells.

Impact of Dkc1 Deficiency on BM Niche Function during Hematopoietic Stem Cell Transplantation in a Conditional Murine Model of X-Linked Dyskeratosis Congenita

Dyskeratosis congenita (DC) is a telomere biology disorder associated with bone marrow failure (BMF) and the triad of dystrophic nails, skin rashes, and leukoplakia. X-linked DC (XDC) is caused by mutations in DKC1, which encodes dyskerin, a nucleolar protein associated with rRNA processing and telomerase complex formation. Hematopoietic stem cell transplantation (HSCT) is currently the only definitive curative treatment for BMF in XDC patients, though many patients exhibit only partial restoration of hematopoietic function. Our previous studies demonstrated that molecular and cellular defects in BM niches contribute to poor donor HSC engraftment in murine models of other BMF syndromes, including Shwachman-Diamond Syndrome (SDS), a disease whose pathogenesis intersects with the role of dyskerin in ribosome biogenesis. These studies led us to hypothesize that BM niche dysfunction contribute to poor graft function in DC patients post-HSCT. Defining specific deficits would enable development of novel targeting strategies to improve HSCT outcomes for patients with XDC. Here, we report that in a novel murine model of XDC driven by conditional Dkc1 deletion, Dkc1 deficiency within mesenchymal BM niche cells causes significant impairment of healthy donor HSC engraftment after HSCT. We have identified defects in multiple pathways within Dkc1-deficient BM niche in response to myeloablative conditioning that may drive this reduction in HSC supportive capacity. To generate a murine model with conditional Dkc1 deletion in BM niche, we crossed Dkc1l/lmice with Mx1 Cre+mice, inducing Cre expression in hematopoietic cells and Mx1-inducible niche cells by polyinosinic-polycytidylic acid administration. At baseline, the resulting Mx1 CreDkc1 Exc mice exhibit mild hematopoietic abnormalities versus Dkc1 l/l controls, including variable thrombocytopenia, increased blood monocytes, and reduced BM lymphoid progenitors. To assess capacity of BM niches from Mx1 CreDkc1 Exc mice to efficiently engraft donor HSC, we transplanted Mx1 CreDkc1 Exc mice and Dkc1 l/l controls with healthy GFP + wild-type donor BM and assessed efficiency of donor HSC engraftment at 2 weeks following HSCT using competitive secondary HSCT. The Mx1 CreDkc1 Exc recipients exhibited significantly impaired efficiency of donor HSC engraftment compared with controls, as indicated by a 25-30% reduction of long-term GFP + cell reconstitution in mature blood lineages in secondary recipients (Figure 1). We next sought to identify molecular and cellular defects in BM niche that contribute to the engraftment deficits in Mx1 CreDkc1 Exc mice. Flow cytometry analysis of BM niche cells 48 hours after total body irradiation (TBI) showed Mx1 CreDkc1 Exc mice exhibited a 20% reduction of CD11b+F4/80+ osteomacs compared to Dkc1 l/l controls. In contrast to our published findings in a model of SDS, the frequencies of osteoprogenitors, mesenchymal stem cell, endothelial cells and megakaryocytes in BM niche were similar between irradiated Dkc1-deficient and control mice. We then performed multiplex ELISA and RNA-seq analyses on isolated BM niche cells from Mx1 CreDkc1 Exc versus control BM at 48 hours post-TBI. Irradiated Mx1 CreDkc1 Exc BM niche demonstrated lower expression of multiple chemokines, including CXCL1, CXCL12, CCL1, CCL11 and CCL12, known to be critical for recruiting HSC and many leukocyte subsets. Dkc1 deficiency in post-TBI BM niche cells also led to lower expression of genes that regulate production of IFN-γ and IL-1, resulting in reduced expression level of both cytokines that may drive decreased osteomacs seen in Dkc1-deficient BM niches. Moreover, Gene Ontology analysis indicates that after TBI, Dkc1 deficiency within BM niches resulted in downregulation of gene expression involved in cell-cell adhesion and extracellular matrix, both of which are critical for HSC enlodgement and engraftment after HSCT. Taken together, our results demonstrated that Dkc1 deficiency in BM niche cells that are not replaced by HSCT impairs the ability of these niches to engraft donor HSC efficiently. Disruptions of molecular and cellular pathways implicated in the niche dysfunction of this XDC model are distinct from our previous findings with SDS model. Ongoing and future studies are needed to define which of these pathways have the highest potential to serve as translational targets to improve HSCT success rate to treat BMF in patients with XDC.

Dysregulated Proteostasis Restrains the Developmental HSC Expansion in Fanconi Anemia

Hematopoietic stem cells (HSCs) require precise regulation of protein synthesis, processing, and degradation. Protein homeostasis (Proteostasis) is particularly critical during development, relying on chaperone proteins to maintain folding capacity in a highly proliferative HSC pool. Even modest changes in synthesis or degradation lead to the accumulation of unfolded proteins and compromise fetal liver HSC pool formation. Fanconi anemia (FA) is a multisystem disorder that manifests with bone marrow failure (BMF) early in life. Previous reports from our lab and others suggest that pre-natal HSC depletion in FA is a critical factor for the early onset of clinical BMF. To identify the cellular and molecular mechanisms underlying prenatal HSC defects in FA, we performed single-cell transcriptome profiling in both Fancd2 +/+and Fancd2 -/- FL samples. Gene set enrichment analysis (GSEA) of differentially expressed genes revealed significantly increased expression of c-Myc target genes in Fancd2 -/ - HSCs (normalized enrichment score, NES: 2.04, p<10 -5 FDR<10 -5), matched by an increase in c-Myc in Fancd2 -/- LT-HSC (CD150 + CD48 - Lineage - Sca-1 + c-KIT +) at the protein level (1.7-fold (p<0.05). Our sc-RNA Seq data further showed an increase in ribosome biogenesis (NES: 1.8, p<10 -5, FDR<10 -3) and translation (NES: 2.2, p<10 -5, FDR<10 -5) in Fancd2 -/- HSC, functionally corroborated by increased protein synthesis rates across Fancd2 -/- FL HSPC subpopulations (1.3-fold in LT- HSCs, p<0.01) using the in vivo O-Propargyl-puromycin (OPP) assay. Not surprisingly, the increased protein synthesis rates exceed the physiologic protein folding capacity, leading to elevated levels of unfolded proteins in Fancd2 -/- FL HSPCs by Tetraphenylethene maleimide (TPE-MI) stain. The aggregated misfolded proteins (aggresomes) in Fancd2 -/- FL HSPCs were also observed by immunofluorescence microscopy. Revisiting our scRNA Seq data, the observed proteotoxic stress was also reflected in the GSEA enrichment for unfolded protein response (UPR; NES: 1.30, p=0.01, FDR=0.01) as well as heat shock stress response (NES:1.7,p<0.0001, FDR=0.09), and coincided with upregulation of response to heat shock factor-1 ( Hsf-1; NES:1.86,p<0.0001, FDR=0.02) in Fancd2 -/- FL HSCs. Because chaperone supplementation has been shown to enhance HSC protein folding capacity, we serially injected the bile acid TUDCA (a chemical chaperone) into the pregnant dam. This not only significantly reduced unfolded protein levels, but indeed rescued FL HSC numbers in Fancd2 -/- littermates. Our observations in aggregate indicate that dysregulated proteostasis is a previously unrecognized pathogenic process constraining fetal Fancd2 -/- hematopoiesis. Protection of the HSPC proteome appears to be a new, non-canonical function of FANCD2, without immediate connection to the FA pathway and DNA crosslink repair. Importantly, we demonstrate the successful pharmacological rescue of the fetal Fancd2 -/- HSPC pool.

Hnrnpk Overexpression Drives Nucleolar Aberrancies Causing Ribosomopathies

Background: Protein biogenesis is a complex process involving nucleoli and ribosomes. Alterations in any step could lead to alterations in ribosome functionality and protein synthesis. Hnrnpk is an RNA-binding protein (RBP) involve in these processes, finding that an overexpression (OE) produces nucleus and nucleolar stress (NS), decreases transcription, and drives an imbalance in ribosome biogenesis, causing a reduced translation. Aims: To elucidate how hnRNP K dysregulation affects the hematopoietic stem cell (HSCs) biology. Methods: To study the impact of Hnrnpk OE in vivo, we developed an inducible tamoxifen mouse model, HnrnpkTg/hUbc-CreERT2. Survival was evaluated by Kaplan-Meier, phenotype was described by symptoms/signs, CBC, bone marrow (BM) H/E, IHC and FCM analysis, and serum IL-6 ELISA. HSCs were cultured to study the impact of Hnrnpk OE in the HSCs dynamics. Hnrnpk OE was established in vitro using CRISPR/SAM. RNA-seq analysis was performed in a single read 85-base format and analyzed with DESeq2. TMT-based deep proteome profiling was also performed. Both were GSEA preranked. Transcription and translation were tested using Click-it RNA and HPG kit respectively, and translation efficiency by polysome assay. NS were analyzed by confocal microscopy and transmission electron microscopy (TEM). Protein-protein interaction between Hnrnpk and Ncl was studied by IP. Possible phenotype rescue was carried using HnrnpkTg/hUbc-CreERT2/ c-Myclox/wt, HnrnpkTg/hUbc-CreERT2/ Tp53lox/wt and HnrnpkTg/hUBC-CreERT2/ NclKDin vitro and in vivo models. Cell cycle FACS, senescence assays and karyotyping were performed. Molecular mechanism was elucidated by qRT-PCR and WB. Results: Hnrnpk Tg/hUbc-CreERT2 mice had widespread Hnrnpk OE and lifespan's reduction. By CBC, we found the development of leukopenia, lymphopenia, anaemia and thrombocytopenia (Fig.A). BM H/E, IHC and FCM showed a reduction of B220 + and CD34 + and Sca1 + HSCs, and an increment in myeloid cells (Fig. B). Also, we found higher senescent β-galactosidase expression in BM and IL-6 in vivo (Fig.C). Then, we found a decay in viability and an exhaustion in HSCs (Fig.D). To understand Hnrnpk implication in BM failure phenotype in vivo, we generated Hnrnpk OE cells (Fig.E). RNA-seq showed an upregulation in G2/M-checkpoint pathway related molecules (Fig.F), confirmed by FACS analysis, showing and increment of arrested G2/M phase-cells (Fig.G). Moreover, we showed a rise in β-galactosidase activity, polyploidy and genomic instability (Fig. H), linked to an increment in p21 and p16 (Fig.I). Then, TEM revealed nucleolar alterations in the in vitro model, including segregation, anormal accumulations or fragmentation of nucleolar components, alterations validated by confocal microscopy (Fig.J-K). Also, we found a Ncl increment in vitro, consistent with the protein-protein interaction between Hnrnpk and Ncl. Then, proteomics showed that Hnrnpk OE correlates with ribosome biogenesis regulators, c-Myc and mTOR dysregulation (Fig.L). Moreover, we found a decrease in transcription (Fig.M), consistent with rRNAs reduction, driving translation and protein synthesis deficiency (Fig.N). There was a partial reversion of NS hallmarks in HnrnpkTg/hUbc-CreERT2/ c-Myclox/wt, HnrnpkTg/hUbc-CreERT2/ Tp53lox/wt and HnrnpkTg/hUBC-CreERT2/ NclKD MEFs in vitro models and partially rescue phenotype in vivo (Fig.O). Finally, we focused on the nucleolus. Thus, Hnrnpk OE cells showed a reduction in Fbl, increase in Ncl and Ncl diffusion in nucleoplasm. All these data suggest the existence of a ribosompathy-like phenotype. Conclusions: this work found that RBPs dysregulation such as Hnrnpk OEdrives BM failure phenotype, promoting the exhaustion of HSCs by nucleoulus/ribosome dysregulation that triggers cell cycle arrest and apoptosis, dependent of p53. We therefore suggest that Hnrnpk induces ribosome dysfunction consistent with some types of ribosomopathies. This work was financially supported by CRIS contra el Cancer Association (NGO) AES ISCIII (PI18/00295, PI21/00191), ISCIII Miguel Servet (CP19/00140), Cancer Research UK [C355/A26819], Sociedad Española de Hematología y Hemoterapia (SEHH), FC AECC and AIRC under the Accelerator Award Program and National Cancer Institutes of Health Award (R01CA207204, SMP) Leukemia and Lymphoma Society (6577-19, SMP).