CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.

Islands of genomic stability in the face of genetically unstable metastatic cancer.

Introduction: Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because ~10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9. Methods: Whole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing. Results: We found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases. Conclusions: Regions of truncal LOH are strongly retained in the presence of genetic instability, and therefore represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer.

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within the noncoding regions. We propose a novel, cancer-specific killing approach using CRISPR-Cas9 which exploits the requirement of a protospacer adjacent motif (PAM) for Cas9 activity. Through whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002), we identified an average of 417 somatic PAMs per tumor produced from single base substitutions. We analyzed 591 paired T-N samples from The International Cancer Genome Consortium and discovered medians of ~455 somatic PAMs per tumor in pancreatic, ~2800 in lung, and ~3200 in esophageal cancer cohorts. Finally, we demonstrated >80% selective cell death of two targeted pancreatic cancer cell lines in co-cultures using 4-9 sgRNAs, targeting noncoding regions, designed from the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs through WGS.

Mechanism of delayed cell death following simultaneous CRISPR-Cas9 targeting in pancreatic cancers.

When we transduced pancreatic cancers with sgRNAs that targeted 2-16 target sites in the human genome, we found that increasing the number of CRISPR-Cas9 target sites produced greater cytotoxicity, with >99% growth inhibition observed by targeting only 12 sites. However, cell death was delayed by 2-3 weeks after sgRNA transduction, in contrast to the repair of double strand DNA breaks (DSBs) that happened within 3 days after transduction. To explain this discrepancy, we used both cytogenetics and whole genome sequencing to interrogate the genome. We first detected chromatid and chromosome breaks, followed by radial formations, dicentric, ring chromosomes, and other chromosomal aberrations that peaked at 14 days after transduction. Structural variants (SVs) were detected at sites that were directly targeted by CRISPR-Cas9, including SVs generated from two sites that were targeted, but the vast majority of SVs (89.4%) were detected elsewhere in the genome that arose later than those directly targeted. Cells also underwent polyploidization that peaked at day 10 as detected by XY FISH assay, and ultimately died via apoptosis. Overall, we found that the simultaneous DSBs induced by CRISPR-Cas9 in pancreatic cancers caused chromosomal instability and polyploidization that ultimately led to delayed cell death.

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate.

BACKGROUND: Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered and exposure-based mouse models in different mouse strains and prostate lobes within each mouse strain, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. METHODS: In this study, we used single-cell RNA-sequencing (scRNA-seq) methods to assess the single-cell transcriptomes of 6-month-old mouse prostates from two commonly used mouse strains, friend virus B/NIH jackson (FVB/NJ) (N = 2) and C57BL/6J (N = 3). For each mouse, the lobes of the prostate were dissected (anterior, dorsal, lateral, and ventral), and individual scRNA-seq libraries were generated. In situ and pathological analyses were used to explore the spatial and anatomical distributions of novel cell types and molecular markers defining these cell types. RESULTS: Data dimensionality reduction and clustering analysis of scRNA-seq data revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Gene set enrichment analysis comparing luminal cells by strain showed enrichment of proto-Oncogene targets in FVB/NJ mice. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. CONCLUSIONS: Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

Androgen receptor activity in prostate cancer dictates efficacy of bipolar androgen therapy through MYC.

Testosterone is the canonical growth factor of prostate cancer but can paradoxically suppress its growth when present at supraphysiological levels. We have previously demonstrated that the cyclical administration of supraphysiological androgen (SPA), termed bipolar androgen therapy (BAT), can result in tumor regression and clinical benefit for patients with castration-resistant prostate cancer. However, predictors and mechanisms of response and resistance have been ill defined. Here, we show that growth inhibition of prostate cancer models by SPA required high androgen receptor (AR) activity and were driven in part by downregulation of MYC. Using matched sequential patient biopsies, we show that high pretreatment AR activity predicted downregulation of MYC, improved clinical response, and prolonged progression-free and overall survival for patients on BAT. BAT induced strong downregulation of AR in all patients, which is shown to be a primary mechanism of acquired resistance to SPA. Acquired resistance was overcome by alternating SPA with the AR inhibitor enzalutamide, which induced adaptive upregulation of AR and resensitized prostate cancer to SPA. This work identifies high AR activity as a predictive biomarker of response to BAT and supports a treatment paradigm for prostate cancer involving alternating between AR inhibition and activation.

Identifying Phased Mutations and Complex Rearrangements in Human Prostate Cancer Cell Lines through Linked-Read Whole-Genome Sequencing.

A limited number of cell lines have fueled the majority of preclinical prostate cancer research, but their genomes remain incompletely characterized. Here, we utilized whole-genome linked-read sequencing for comprehensive characterization of phased mutations and rearrangements in the most commonly used cell lines in prostate cancer research including PC3, LNCaP, DU145, CWR22Rv1, VCaP, LAPC4, MDA-PCa-2b, RWPE-1, and four derivative castrate-resistant (CR) cell lines LNCaP_Abl, LNCaP_C42b, VCaP-CR, and LAPC4-CR. Phasing of mutations allowed determination of "gene-level haplotype" to assess whether genes harbored heterozygous mutations in one or both alleles. Phased structural variant analysis allowed identification of complex rearrangement chains consistent with chromothripsis and chromoplexy. In addition, comparison of parental and derivative CR lines revealed previously known and novel genomic alterations associated with the CR phenotype. IMPLICATIONS: This study therefore comprehensively characterized phased genomic alterations in the commonly used prostate cancer cell lines, providing a useful resource for future prostate cancer research.

Entinostat Decreases Immune Suppression to Promote Antitumor Responses in a HER2+ Breast Tumor Microenvironment.

Therapeutic combinations to alter immunosuppressive, solid tumor microenvironments (TME), such as in breast cancer, are essential to improve responses to immune checkpoint inhibitors (ICI). Entinostat, an oral histone deacetylase inhibitor, has been shown to improve responses to ICIs in various tumor models with immunosuppressive TMEs. The precise and comprehensive alterations to the TME induced by entinostat remain unknown. Here, we employed single-cell RNA sequencing on HER2-overexpressing breast tumors from mice treated with entinostat and ICIs to fully characterize changes across multiple cell types within the TME. This analysis demonstrates that treatment with entinostat induced a shift from a protumor to an antitumor TME signature, characterized predominantly by changes in myeloid cells. We confirmed myeloid-derived suppressor cells (MDSC) within entinostat-treated tumors associated with a less suppressive granulocytic (G)-MDSC phenotype and exhibited altered suppressive signaling that involved the NFκB and STAT3 pathways. In addition to MDSCs, tumor-associated macrophages were epigenetically reprogrammed from a protumor M2-like phenotype toward an antitumor M1-like phenotype, which may be contributing to a more sensitized TME. Overall, our in-depth analysis suggests that entinostat-induced changes on multiple myeloid cell types reduce immunosuppression and increase antitumor responses, which, in turn, improve sensitivity to ICIs. Sensitization of the TME by entinostat could ultimately broaden the population of patients with breast cancer who could benefit from ICIs.

Comparative genomics of tadpole shrimps (Crustacea, Branchiopoda, Notostraca): Dynamic genome evolution against the backdrop of morphological stasis.

This analysis presents five genome assemblies of four Notostraca taxa. Notostraca origin dates to the Permian/Upper Devonian and the extant forms show a striking morphological similarity to fossil taxa. The comparison of sequenced genomes with other Branchiopoda genomes shows that, despite the morphological stasis, Notostraca share a dynamic genome evolution with high turnover for gene families' expansion/contraction and a transposable elements content comparable to other branchiopods. While Notostraca substitutions rate appears similar or lower in comparison to other branchiopods, a subset of genes shows a faster evolutionary pace, highlighting the difficulty of generalizing about genomic stasis versus dynamism. Moreover, we found that the variation of Triops cancriformis transposable elements content appeared linked to reproductive strategies, in line with theoretical expectations. Overall, besides providing new genomic resources for the study of these organisms, which appear relevant for their ecology and evolution, we also confirmed the decoupling of morphological and molecular evolution.

Decreased Activated CD4+ T Cell Repertoire Diversity After Antiretroviral Therapy in HIV-1/HCV Coinfection Correlates with CD4+ T Cell Recovery.

Dysfunctional immune activation accumulates during chronic viral infection and contributes to disease pathogenesis. In HIV-1, immune activation is exacerbated by concurrent infection with hepatitis C virus (HCV), accelerating depletion of CD4+ T cells. HIV-1 suppression with antiretroviral therapy (ART) generally reconstitutes CD4+ T cell counts, while also reducing the proportion that is activated. Whether this immune reconstitution also reduces the complexity of the CD4+ T cell population is unknown. We sought to characterize the relationship between activated CD4+ T cell repertoire diversity and immune reconstitution following ART in HIV-1/HCV coinfection. We extracted T cell receptor (TCR) sequences from RNA sequencing data obtained from activated CD4+ T cells of HIV-1/HCV coinfected individuals before and after treatment with ART (clinical trial NCT01285050). There was notable heterogeneity in both the extent of CD4+ T cell reconstitution and in the change in activated CD4+ TCR repertoire diversity following ART. Decreases in activated CD4+ TCR repertoire diversity following ART were predictive of the degree of CD4+ T cell reconstitution. The association of decreased activated CD4+ TCR repertoire diversity and improved CD4+ T cell reconstitution may represent loss of nonspecifically activated TCR clonotypes, and possibly selective expansion of specifically activated CD4+ clones. These results provide insight into the dynamic relationship between activated CD4+ TCR diversity and CD4+ T cell recovery of HIV-1/HCV coinfected individuals after suppression of HIV-1 viremia.

Transcriptional programs of neoantigen-specific TIL in anti-PD-1-treated lung cancers.

PD-1 blockade unleashes CD8 T cells1, including those specific for mutation-associated neoantigens (MANA), but factors in the tumour microenvironment can inhibit these T cell responses. Single-cell transcriptomics have revealed global T cell dysfunction programs in tumour-infiltrating lymphocytes (TIL). However, the majority of TIL do not recognize tumour antigens2, and little is known about transcriptional programs of MANA-specific TIL. Here, we identify MANA-specific T cell clones using the MANA functional expansion of specific T cells assay3 in neoadjuvant anti-PD-1-treated non-small cell lung cancers (NSCLC). We use their T cell receptors as a 'barcode' to track and analyse their transcriptional programs in the tumour microenvironment using coupled single-cell RNA sequencing and T cell receptor sequencing. We find both MANA- and virus-specific clones in TIL, regardless of response, and MANA-, influenza- and Epstein-Barr virus-specific TIL each have unique transcriptional programs. Despite exposure to cognate antigen, MANA-specific TIL express an incompletely activated cytolytic program. MANA-specific CD8 T cells have hallmark transcriptional programs of tissue-resident memory (TRM) cells, but low levels of interleukin-7 receptor (IL-7R) and are functionally less responsive to interleukin-7 (IL-7) compared with influenza-specific TRM cells. Compared with those from responding tumours, MANA-specific clones from non-responding tumours express T cell receptors with markedly lower ligand-dependent signalling, are largely confined to HOBIThigh TRM subsets, and coordinately upregulate checkpoints, killer inhibitory receptors and inhibitors of T cell activation. These findings provide important insights for overcoming resistance to PD-1 blockade.

A conserved intratumoral regulatory T cell signature identifies 4-1BB as a pan-cancer target.

Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB-expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB-expressing Tregs represents a strategy with potential activity across cancer types.

Mitochondrial genome diversity and evolution in Branchiopoda (Crustacea).

BACKGROUND: The crustacean class Branchiopoda includes fairy shrimps, clam shrimps, tadpole shrimps, and water fleas. Branchiopods, which are well known for their great variety of reproductive strategies, date back to the Cambrian and extant taxa can be mainly found in freshwater habitats, also including ephemeral ponds. Mitochondrial genomes of the notostracan taxa Lepidurus apus lubbocki (Italy), L. arcticus (Iceland) and Triops cancriformis (an Italian and a Spanish population) are here characterized for the first time and analyzed together with available branchiopod mitogenomes. RESULTS: Overall, branchiopod mitogenomes share the basic structure congruent with the ancestral Pancrustacea model. On the other hand, rearrangements involving tRNAs and the control region are observed among analyzed taxa. Remarkably, an unassigned region in the L. apus lubbocki mitogenome showed a chimeric structure, likely resulting from a non-homologous recombination event between the two flanking trnC and trnY genes. Notably, Anostraca and Onychocaudata mitogenomes showed increased GC content compared to both Notostraca and the common ancestor, and a significantly higher substitution rate, which does not correlate with selective pressures, as suggested by dN/dS values. CONCLUSIONS: Branchiopod mitogenomes appear rather well-conserved, although gene rearrangements have occurred. For the first time, it is reported a putative non-homologous recombination event involving a mitogenome, which produced a pseudogenic tRNA sequence. In addition, in line with data in the literature, we explain the higher substitution rate of Anostraca and Onychocaudata with the inferred GC substitution bias that occurred during their evolution.

CMPK2 and BCL-G are associated with type 1 interferon-induced HIV restriction in humans.

Type 1 interferons (IFN) are critical for host control of HIV and simian immunodeficiency virus. However, it is unknown which of the hundreds of interferon-stimulated genes (ISGs) restrict HIV in vivo. We sequenced RNA from cells that support HIV replication (activated CD4+ T cells) in 19 HIV-infected people before and after interferon-α2b (IFN-α2b) injection. IFN-α2b administration reduced plasma HIV RNA and induced mRNA expression in activated CD4+ T cells: The IFN-α2b-induced change of each mRNA was compared to the change in plasma HIV RNA. Of 99 ISGs, 13 were associated in magnitude with plasma HIV RNA decline. In addition to well-known restriction factors among the 13 ISGs, two novel genes, CMPK2 and BCL-G, were identified and confirmed for their ability to restrict HIV in vitro: The effect of IFN on HIV restriction in culture was attenuated with RNA interference to CMPK2, and overexpression of BCL-G diminished HIV replication. These studies reveal novel antiviral molecules that are linked with IFN-mediated restriction of HIV in humans.

Integrated Analysis of Whole-Genome ChIP-Seq and RNA-Seq Data of Primary Head and Neck Tumor Samples Associates HPV Integration Sites with Open Chromatin Marks.

Chromatin alterations mediate mutations and gene expression changes in cancer. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) has been utilized to study genome-wide chromatin structure in human cancer cell lines, yet numerous technical challenges limit comparable analyses in primary tumors. Here we have developed a new whole-genome analytic pipeline to optimize ChIP-Seq protocols on patient-derived xenografts from human papillomavirus-related (HPV+) head and neck squamous cell carcinoma (HNSCC) samples. We further associated chromatin aberrations with gene expression changes from a larger cohort of the tumor and normal samples with RNA-Seq data. We detect differential histone enrichment associated with tumor-specific gene expression variation, sites of HPV integration in the human genome, and HPV-associated histone enrichment sites upstream of cancer driver genes, which play central roles in cancer-associated pathways. These comprehensive analyses enable unprecedented characterization of the complex network of molecular changes resulting from chromatin alterations that drive HPV-related tumorigenesis. Cancer Res; 77(23); 6538-50. ©2017 AACR.

Intracellular HIV-1 RNA and CD4+ T-cell activation in patients starting antiretrovirals.

OBJECTIVE: To assess if the reduction in HIV-1 RNA in CD4 T cells is correlated with the persistence of immune activation following early antiretroviral therapy (ART). DESIGN: Clinical trial (NCT01285050). METHODS: Next-generation sequencing was used to study total RNA from activated CD4 T cells (CD38 and human leukocyte antigen - antigen D related (HLA-DR) expressing) collected from 19 treatment-naïve HIV-1/hepatitis C virus-infected patients before and early after ART initiation (≥12 weeks after plasma HIV-1 RNA <50 copies/ml). To validate comparisons, pre and post-ART measures were adjusted for input RNA and overall read number. RESULTS: As expected, ART use was associated with a median [interquartile range (IQR)] 4.3% (2.2-8.3) reduction in the proportion of activated CD4 T cells (P = 0.0008). Whereas in those activated CD4 T cells no consistent differences in overall gene expression were detected, interferon-stimulated gene expression declined (P < 2 × 10). Pre-ART, sorted activated CD4 T cells contained a median (IQR) of 959 (252-1614) HIV-1 reads/10 reads compared with 72 (55-152) HIV-1 reads/10 reads after at least 12 weeks of suppressive ART (P = 8 × 10). The decrease in HIV-1 reads in activated CD4 T cells was associated with the change in plasma HIV-1 RNA levels (r = 0.77, P = 2 × 10) and the change in the proportion of activated CD4 T cells (r = 0.70, P = 0.0016). CONCLUSION: Months of ART led to a marked decrease in cell-associated HIV-1 RNA and interferon-stimulated genes expression in activated CD4 T cells that were strongly associated with the reduction in the proportion of activated CD4 T cells.

NUP98-PHF23 fusion is recurrent in acute myeloid leukemia and shares gene expression signature of leukemic stem cells.

Chromosome translocations involving nucleoporin 98 gene (NUP98) have been identified in a wide array of hematologic malignancies, and the resulting NUP98-associated fusions are known to play a critical role in leukemogensis through dysregulation of gene expression. Although NUP98-associated fusions were initially thought to be rare, application of molecular technologies has revealed that cryptic translocations involving NUP98 are more frequent than previously appreciated. We report an additional case of t(11;17)(p15;p13) resulting in the fusion of NUP98 and plant homeodomain finger 23 (PHF23) in a pediatric patient with acute myeloid leukemia (AML). Using RNA sequencing, we determined in-frame fusion points and also analyzed the gene expression profile of NUP98-PHF23 positive AML. Gene set enrichment analysis (GSEA) demonstrates that NUP98-PHF23 fusion shares gene expression signature of NUP98-HOXA9 fusion, the prototype of the NUP98-associated fusions, as well as the signature of leukemic stem cells. To our knowledge this is the first transcriptome analysis of human samples with NUP98-PHF23 positive AML. Our findings are in support of the gene expression study of NUP98-PHF23 mouse model and validate the usefulness of the mouse model in developing therapeutic strategies for the treatment of subsets of AML.