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Purpose: Antiendocrine therapy remains the most effective treatment for estrogen receptor–positive (ER+) breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer–associated fibroblasts (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance.Experimental Design: Tissues from patients with ER+ breast cancer were analyzed for the presence of CD146-positive (CD146pos) and CD146-negative (CD146neg) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells cocultured with CD146pos or CD146neg fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER+ breast cancer.Results: We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes.Conclusions: Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer and should be considered a target for drug development. Clin Cancer Res; 23(7); 1710–21. ©2016 AACR.

SFig. 1: Cellular morphology defines tumor cell from stroma.; SFig. 2: Primary derived stroma from cancer-associated and normal breast tissue is highly enriched for fibroblasts.; SFig 3: Immortalized primary derived fibroblasts express high levels of vimentin and FSP1.; SFig. 4: Primary derived cancer fibroblasts are highly similar to normal bone marrow-derived fibroblasts.; SFig. 5: Influence from CD146neg fibroblasts on ER+ breast cancer cells reduces ER expression and estrogen dependency.; SFig 6. Influence from CD146neg fibroblasts on ER+ breast cancer is predictive of decreased recurrence-free survival.

Tamoxifen resistant genes up-regulated in ER+ breast cancer cells influenced by CD146neg fibroblasts.

Supplemental Figure Legends.

Tamoxifen responsive genes up-regulated in ER+ breast cancer cells influenced by CD146pos fibroblasts.

Supplemental method section with increased detail regarding methods used in the manuscript.

Expression of genes associated with activated fibroblasts.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human disease remains unexplored. Here, we investigated the impact of non-synonymous mutations in SF3B1 and U2AF1, two commonly mutated splicing factors in cancer, on transcription. We find that the mutations impair RNA Polymerase II (RNAPII) transcription elongation along gene bodies leading to transcription-replication conflicts, replication stress and altered chromatin organization. This elongation defect is linked to disrupted pre-spliceosome assembly due to impaired association of HTATSF1 with mutant SF3B1. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC complex, which, when modulated, normalize transcription defects and their downstream effects. Our findings shed light on the mechanisms by which oncogenic mutant spliceosomes impact chromatin organization through their effects on RNAPII transcription elongation and present a rationale for targeting the Sin3/HDAC complex as a potential therapeutic strategy.GRAPHICAL ABSTRACTHIGHLIGHTS-Oncogenic mutations of SF3B1 and U2AF1 cause a gene-body RNAPII elongation defect-RNAPII transcription elongation defect leads to transcription replication conflicts, DNA damage response, and changes to chromatin organization and H3K4me3 marks-The transcription elongation defect is linked to disruption of the early spliceosome formation through impaired interaction of HTATSF1 with mutant SF3B1.-Changes to chromatin organization reveal potential therapeutic strategies by targeting the Sin3/HDAC pathway

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3′ untranslated regions (3′UTRs) of mRNA, but the identity of functional targets remains unknown. Here, we used individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of mRNAs largely in 3′UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more ‘UAG’ motifs compared to targets bound nonproductively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute loss. Using complementary approaches, we determined eukaryotic translation initiation factor 3A (EIF3A) to be an immediate, direct target. We propose that MSI2 downregulation of EIF3A amplifies these effects on translation. Our results also underscore the challenges in defining functional targets of RBPs since mere binding does not imply a discernible functional interaction.

Mutations in the tumor suppressor gene TP53 are detected in 5–10% of patients with acute myeloid leukemia (AML) and myelodysplastic syndromes. TP53 mutations have been associated with complex karyotypes, therapy-related malignancies, lower response rates to cytotoxic chemotherapy, and an overall adverse prognosis. In this single-center retrospective study, we analyzed the clinicopathologic characteristics and outcomes of 83 patients with TP53-mutated myeloid malignancies treated at Yale Cancer Center between 9/2015 and 5/2019. Complex karyotypes (n=75; 90%) and therapy-related malignancies (n=32; 39%) were common. Median overall survival (OS) was 7.6 months. Intensive chemotherapy did not improve OS compared to lower-intensity treatment for AML patients. Patients who underwent allogeneic hematopoietic stem cell transplant (alloHSCT) had a significantly longer median OS, despite relatively limited follow-up. In conclusion, our data confirm the limited efficacy of intensive chemotherapy approaches for TP53-mutated patients with myeloid neoplasms and suggest that a minority of patients achieve long-term survival with alloHSCT.

Scalable isogenic models of cancer-associated mutations are critical to studying dysregulated gene function. Nonsynonymous mutations of splicing factors, which typically affect one allele, are common in many cancers, but paradoxically confer growth disadvantage to cell lines, making their generation and expansion challenging. Here, we combine AAV-intron trap, CRISPR/Cas9, and inducible Cre-recombinase systems to achieve >90% efficiency to introduce the oncogenic K700E mutation in SF3B1, a splicing factor commonly mutated in multiple cancers. The intron-trap design of AAV vector limits editing to one allele. CRISPR/Cas9-induced double stranded DNA breaks direct homologous recombination to the desired genomic locus. Inducible Cre-recombinase allows for the expansion of cells prior to loxp excision and expression of the mutant allele. Importantly, AAV or CRISPR/Cas9 alone results in much lower editing efficiency and the edited cells do not expand due to toxicity of SF3B1-K700E. Our approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes confer a growth disadvantage., Boddu et al combined AAV-intron trap, CRISPR/Cas9 and inducible Cre-recombinase systems to introduce the oncogenic K700E mutation in SF3B1, which is a splicing factor commonly mutated in multiple cancers, with more than 90% efficiency. Their approach can be readily adapted to generate scalable isogenic systems where mutant oncogenes are detrimental to cell culture growth.

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Cohesin is a ring complex composed of four core subunits and seven regulatory subunits. In an effort to comprehensively identify additional cohesin-interacting proteins, we used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in cultured human cells, and we performed MS analyses on dual-affinity purifications. In addition to reciprocally identifying all known components of cohesin, we found that cohesin interacts with a panoply of splicing factors and RNA-binding proteins (RBPs). These included diverse components of the U4/U6.U5 tri-small nuclear ribonucleoprotein complex and several splicing factors that are commonly mutated in cancer. The interaction between cohesin and splicing factors/RBPs was RNA- and DNA-independent, occurred in chromatin, was enhanced during mitosis, and required RAD21. Furthermore, cohesin-interacting splicing factors and RBPs followed the cohesin cycle and prophase pathway of cell cycle–regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors and RBPs resulted in aberrant mitotic progression. These results provide a comprehensive view of the endogenous human cohesin interactome and identify splicing factors and RBPs as functionally significant cohesin-interacting proteins.

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate decisions in normal and cancer stem cells. MSI2 appears to repress translation by binding to 3’ untranslated regions (3’UTRs) of mRNA, but the identity of functional targets remains unknown. Here we used iCLIP to identify direct RNA binding partners of MSI2 and integrated these data with polysome profiling to obtain insights into MSI2 function. iCLIP revealed specific MSI2 binding to thousands of target mRNAs largely in 3’UTRs, but translational differences were restricted to a small fraction of these transcripts, indicating that MSI2 regulation is not triggered by simple binding. Instead, the functional targets identified here were bound at higher density and contain more “U/TAG” motifs compared to targets bound non-productively. To further distinguish direct and indirect targets, MSI2 was acutely depleted. Surprisingly, only 50 transcripts were found to undergo translational induction on acute MSI2 loss. Eukaryotic elongation factor 3A (EIF3A) was determined to be an immediate, direct target. We propose that MSI2 down-regulation of EIF3A amplifies these effects on the proteome. Our results also underscore the challenges in defining functional targets of RBP since mere binding does not imply a discernible functional interaction.

Simple Summary Triple negative breast cancer is a disease with limited treatment options and the poorest outcome across all breast cancer subtypes, thus the need for new effective therapies is high. We recently found that navitoclax displays synergistic anti-proliferative and apoptotic activities with other drugs in treatment of triple negative breast cancer cells, but the resistance to treatment is still a limiting factor. Therefore, we investigated the effects of navitoclax treatment on the transcriptome, genome and epigenome in vitro to better understand the process of developing resistance. We discovered and validated a list of multiple, previously unknown markers of drug resistance that can help in patient selection in future clinical trials involving navitoclax. Abstract Cancer cells employ various defense mechanisms against drug-induced cell death. Investigating multi-omics landscapes of cancer cells before and after treatment can reveal resistance mechanisms and inform new therapeutic strategies. We assessed the effects of navitoclax, a BCL2 family inhibitor, on the transcriptome, methylome, chromatin structure, and copy number variations of MDA-MB-231 triple-negative breast cancer (TNBC) cells. Cells were sampled before treatment, at 72 h of exposure, and after 10-day drug-free recovery from treatment. We observed transient alterations in the expression of stress response genes that were accompanied by corresponding changes in chromatin accessibility. Most of these changes returned to baseline after the recovery period. We also detected lasting alterations in methylation states and genome structure that suggest permanent changes in cell population composition. Using single-cell analyses, we identified 2350 genes significantly upregulated in navitoclax-resistant cells and derived an 18-gene navitoclax resistance signature. We assessed the navitoclax-response-predictive function of this signature in four additional TNBC cell lines in vitro and in silico in 619 cell lines treated with 251 different drugs. We observed a drug-specific predictive value in both experiments, suggesting that this signature could help guiding clinical biomarker studies involving navitoclax.

Cancer-associated mutations of the core splicing factor 3 B1 (SF3B1) result in selection of novel 3′ splice sites (3′SS), but precise molecular mechanisms of oncogenesis remain unclear. SF3B1 stabilizes the interaction between U2 snRNP and branch point (BP) on the pre-mRNA. It has hence been speculated that a change in BP selection is the basis for novel 3′SS selection. Direct quantitative determination of BP utilization is however technically challenging. To define BP utilization by SF3B1-mutant spliceosomes, we used an overexpression approach in human cells as well as a complementary strategy using isogenic murine embryonic stem cells with monoallelic K700E mutations constructed via CRISPR/Cas9-based genome editing and a dual vector homology-directed repair methodology. A synthetic minigene library with degenerate regions in 3′ intronic regions (3.4 million individual minigenes) was used to compare BP usage of SF3B1K700E and SF3B1WT. Using this model, we show that SF3B1K700E spliceosomes utilize non-canonical sequence variants (at position −1 relative to BP adenosine) more frequently than wild-type spliceosomes. These predictions were confirmed using minigene splicing assays. Our results suggest a model of BP utilization by mutant SF3B1 wherein it is able to utilize non-consensus alternative BP sequences by stabilizing weaker U2-BP interactions.

Splicing factor 3B1 (SF3B1) is a core splicing protein that stabilizes the interaction between the U2 snRNA and the branch point in the mRNA target during splicing. SF3B1 is heavily phosphorylated at its N terminus and a substrate of cyclin-dependent kinases (CDKs). Although SF3B1 phosphorylation coincides with splicing catalysis, the functional significance of SF3B1 phosphorylation is largely undefined. Here, we show that SF3B1 phosphorylation follows a dynamic pattern during cell cycle progression that depends on CDK activity. SF3B1 is known to interact with chromatin, and we found that SF3B1 maximally interacts with nucleosomes during G(1)/S and that this interaction requires CDK2 activity. In contrast, SF3B1 disassociates from nucleosomes at G(2)/M, coinciding with a peak in CDK1-mediated SF3B1 phosphorylation. Thus, CDK1 and CDK2 appear to have opposing roles in regulating SF3B1–nucleosome interactions. Importantly, these interactions were modified by the presence and phosphorylation status of linker histone H1, particularly the H1.4 isoform. Performing genome-wide analysis of SF3B1–chromatin binding in synchronized cells, we observed that SF3B1 preferentially bound exons. Differences in SF3B1 chromatin binding to specific sites, however, did not correlate with changes in RNA splicing, suggesting that the SF3B1–nucleosome interaction does not determine cell cycle–dependent changes to mRNA splicing. Our results define a cell cycle stage–specific interaction between SF3B1 and nucleosomes that is mediated by histone H1 and depends on SF3B1 phosphorylation. Importantly, this interaction does not seem to be related to SF3B1's splicing function and, rather, points toward its potential role as a chromatin modifier.

Background: Fanconi anemia (FA) is a genetic disease characterized by bone marrow failure, developmental defects, and higher risk of cancer. Mutations in FA genes have been detected commonly in a large swath of cancers. In the FA DNA repair pathway, DNA damage induces the mono-ubiquitination of the FANCI-FANCD2 (ID2) heterodimer and this regulation licenses the execution of downstream DNA damage signaling and repair steps. In response to replication stress, FANCD2 also prevents replication fork collapse during S phase. Bloom syndrome (BS) is also a genomic instability disease, characterized by growth abnormalities and cancer predisposition. The single BS protein, BLM helicase, participates in DNA repair by promoting DNA end resection and double Holliday junction dissolution. It has been shown that BLM is involved in restart of stalled replication fork. FA and BS have functional interactions. In tumor DNA sequencing of the Yale Precision Tumor board, we identified a somatic 6 amino acid deletion in FANCD2 in a head and neck tumor, while a germline point mutation was found on the other allele. We have identified a FANCD2-L822A mutant with defective BLM binding, which was used to further investigate the role of FANCD2-BLM interaction in genome stability and DNA repair. Methods: Highly purified proteins were used to investigate how ID2 affects helicase and DNA end resection activity of the BLM complex. HeLa, FANCD2-deficient, and FANCD2 corrected fibroblast cell lines were used to examine pRPA2 and RAD51 foci formation. We also used DNA fiber assay to detect end resection and isolation of proteins on nascent DNA (iPOND) assay to examine the RAD51 recruitment on replication fork. Results: A somatic 6 amino acid deletion (p819-824) in FANCD2 was identified in a head and neck tumor. FA-D2 mutant cells expressing the mutant cDNA demonstrated defects in FANCD2 mono-ubiquitination and DNA damage hypersensitivity. A FANCD2-L822A mutant with defective BLM binding was identified (Figure A, B). We found that Bloom helicase and its DNA end resection activity within BLM-DNA2-RPA were negatively regulated by the heterodimer ID2 (Figure C, D). Both DNA and BLM binding of the ID2 are required for the inhibitory function. The premature DNA end resection and HU sensitivity in FANCD2 deficient and mutant cells are rescued by BLM knockdown. By iPOND assay, we discovered that FANCD2 antagonizes BLM to promote RAD51 recruitment on HU-stalled replication fork. Conclusions: Our study suggests that the DNA end resection activity of BLM-DNA2 is tightly regulated by FANCD2 to ensure that the nuclease DNA2 normally resects the DNA intermediate needed for efficient DNA repair and RAD51 recruitment to protect replication forks. Our findings highlight that ID2-BLM interaction functions in DNA damage repair to maintain genome stability. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Disruption of posttranscriptional gene regulation is a critical step in oncogenesis that can be difficult to observe using traditional molecular techniques. To overcome this limitation, a modified polyadenylation site sequencing (PAS-seq) protocol was used to generate a genome-wide map of alternative polyadenylation (APA) events in human primary breast tumor specimens and matched normal tissue. This approach identified an APA event in the PRELID1 mRNA that enhances its steady-state level and translational efficiency, and is a strong breast cancer subtype-dependent predictor of patient clinical outcomes. Furthermore, it has been demonstrated that PRELID1 regulates stress response and mitochondrial reactive oxygen species (ROS) production in a cell type–specific manner. Modulation of PRELID1 expression, including its posttranscriptional control, appears to be a common stress response across different cancer types. These data reveal that PRELID1 mRNA processing is an important regulator of cell type–specific responses to stress used by multiple cancers and is associated with patient outcomes. Implications: This study suggests that the regulation of PRELID1 expression, by APA and other mechanisms, plays a role in mitochondrial ROS signaling and represents a novel prognostic factor and therapeutic target in cancer. Mol Cancer Res; 15(12); 1741–51. ©2017 AACR.

IMPACT: We describe a novel methodology that combines CRISPR/Cas9-induced double stranded DNA breaks with homology dependent repair from an adeno associated virus (AAV) encoded template to generate single-allele edited isogenic cell line models of cancer-associated mutations with high efficiency. OBJECTIVES/GOALS: Conventional approaches to creating isogenic knock-ins, to model disease-associated mutations, are limited by poor efficiency and loss of the mutant allele on extended culture. We present an optimized editing approach combining CRISPR/Cas9 with Adeno-associated virus, using mutant SF3B1 as a prototype. METHODS/STUDY POPULATION: Left and right homology arms for SF3B1 were PCR amplified and cloned into pAAV-SEPT-Acceptor plasmid (containing a chimeric intron, neomycin resistance cassette and polyA tail). The disease-associated K700E mutation was introduced by site-directed mutagenesis. Single guide RNA (sgRNA) complexed with recombinant Cas9 along with the AAV donor were delivered into K562 cells, G418 resistant clones selected, and screened for integration by PCR. Confirmed clones were then transduced with a doxycycline-inducible Cre-recombinase containing lentiviral vector. Inducible expression of Cre-recombinase and expression of the mutant allele were confirmed by Western blot and Sanger sequencing respectively. RESULTS/ANTICIPATED RESULTS: Targeted-integration efficiencies among the Neo-resistant clones, generated by AAV-alone and AAV+CRISPR/Cas9, were 16% and 94%, respectively. Single cell cloning after Cre-mediated excision of loxp was unsuccessful presumably due to toxicity of the K700E mutation. To overcome this limitation, clones were transduced with doxycycline-inducible Cre-recombinase lentiviral vector. Doxycycline induction of Cre-recombinase resulted in reliable excision of the loxp cassette and expression of mutant allele at about 50% variable allele frequency (as determined by Sanger sequencing). The approach was validated in additional cell lines and for introduction of N-terminal FLAG tag for SF3B1. DISCUSSION/SIGNIFICANCE OF FINDINGS: Combining AAV and CRISPR/Cas9 can generate scalable single-dominant allele mutants with high precision and efficiency compared to AAV or CRISPR alone. Together with inducible Cre-recombinase, our approach can generate isogenic models where the mutation confers a growth disadvantage.

Recurrent mutations in core splicing factors have been reported in several clonal disorders, including cancers. Mutations in SF3B1, a component of the U2 splicing complex, are the most common. SF3B1 mutations are associated with aberrant pre-mRNA splicing using cryptic 3' splice sites (3'SSs), but the mechanism of their selection is not clear. To understand how cryptic 3'SSs are selected, we performed comprehensive analysis of transcriptome-wide changes to splicing and gene expression associated with SF3B1 mutations in patient samples as well as an experimental model of inducible expression. Hundreds of cryptic 3'SS were detectable across the genome in cells expressing mutant SF3B1. These 3'SS are typically sequestered within RNA secondary structures and poorly accessible compared with their corresponding canonical 3'SS. We hypothesized that these cryptic 3'SS are inaccessible during normal splicing catalysis and that this constraint is overcome in spliceosomes containing mutant SF3B1. This model of secondary structure-dependent selection of cryptic 3'SS was found across multiple clonal processes associated with SF3B1 mutations (myelodysplastic syndrome and chronic lymphocytic leukemia). We validated our model predictions in mini-gene splicing assays. Additionally, we found deregulated expression of proteins with relevant functions in splicing factor-related diseases both in association with aberrant splicing and without corresponding splicing changes. Our results show that SF3B1 mutations are associated with a distinct splicing program shared across multiple clonal processes and define a biochemical mechanism for altered 3'SS choice.

BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA. METHODS. Cells: FA-D2 mutant (PD20) and corrected (D2), HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation was performed using SRSF1 purified from bacteria and FANCD2-FANCI purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay and S9.6 immunofluorescence. RESULTS. In order to study the involvement of splicing factors in the FA pathway, we knocked down SRSF1 by using siRNA. SRSF1 knockdown lead to impaired FANCD2 monoubiquitination after DNA damage as well as diminished cell survival, thus creating a FA-like phenotype (A). In addition, immunoprecipitation in cells (B) and in vitro showed direct interaction between FANCD2 and SRSF1. We found that double FANCD2+SRSF1 knockdown led to increased R-loops compared to FANCD2/SRSF1 single knockdown visualized by S9.6 immunofluorescence. In addition, DART assay revealed colocalization of SRSF1 and FANCD2 with R-loops in highly transcribed genomic sites, known to be R-loop hotspots (C). Finally, we found that SRSF1 interacts with the mRNA export factor NFX1 in a FANCD2-dependent fashion (D) through RNA, suggesting that they could cooperate in the prevention of R-loop formation by exporting accumulated RNA in the nucleus. CONCLUSION. Our findings suggest a novel non-canonical mechanism of FANCD2 via regulation of SRSF1 in R-loop prevention via facilitation of mRNA export, thus preventing genomic instability (E). CLINICAL APPLICABILITY. These results will reveal a novel mechanism for a non-canonical role of the FA pathway in prevention of genomic instability and will shed light upon the most basic cellular processes of the FA pathway, which is germane for delineating more generalized mechanisms of oncogenesis and genome maintenance. As accumulation of R-loops is associated with FA, we propose that R-loops and R-loop processing proteins (such as SRSF1) offer new avenues as relevant therapeutic targets not only for FA but also for multiple cancers. Disclosures No relevant conflicts of interest to declare.

Cancer cells possess various anti-apoptotic defense mechanisms that can protect them from drug induced cell death. We investigated the effects of treatment with the bcl-2/bcl-XL inhibitor Navitoclax on transcriptome, methylome, and genome of MDA-MB-231 cells. Two biological replicates were treated simultaneously and profiled using 10x Genomics single-cell RNA sequencing (scRNAseq; gene expression), bulk RNA sequencing (bRNAseq; gene expression), ATAC sequencing (ATAC; chromatin accessibility), bisulphite targeted sequencing (DNAm; DNA methylation) and shallow whole-genome sequencing (WGS; copy number variants) at baseline before treatment, after 3 days of 10µM navitoclax treatment, and after 10 days of cell recovery from treatment. Variance between biological replicates on transcriptome level was minimal before the treatment (R>0.99), but we noticed divergence in gene expression after recovery from the treatment (both on bulk and single-cell level), mostly driven by cell cycle genes. On other platforms, biological replicates were similar in all 3 phases. After treatment, we observed more genes with decreased expression (n=151) in comparison to baseline, but after 10 days of recovery there were more up-regulated genes (n=655) in comparison to samples after treatment. Chromatin accessibility for most genomic regions increased after treatment in comparison to baseline, suggesting an acute response to the stress caused by drug treatment, and then returned to baseline level after recovery period. DNA methylation patterns showed more regions with decreased methylation after treatment (n=529) in comparison to baseline which remained detectable even after the recovery period. In WGS data, we found 752 genes with deletions and amplifications only in baseline samples representing genomic background of drug sensitive cells, mostly enriched in epithelial mesenchymal transition pathway. On single-cell level, we identified a subset of cells that were resistant to treatment and discovered 2,324 genes significantly up-regulated in these cells, that could be potential markers of resistance. In gene set analysis, these markers were enriched in MYC and E2F target gene sets, and were involved in angiogenesis and JAK-STAT pathway. We measured the expression of 16 top up-regulated markers of resistance in 4 different TNBC cell lines using qPCR and found that 5 were significantly enriched after treatment in 3 cell lines, 5 in 2 cell lines and 5 in a single cell line. Summarizing, we thoroughly described molecular effects of Navitoclax treatment and showed that most cells return to the basal transcriptional state after the drug recovery period, but bulk genome and methylome are permanently changed. Finally, we provided a list of new markers of resistance that may be useful in the studies of combinational therapies with other drugs. This work was funded by Breast Cancer Research Foundation. Citation Format: Michal Marczyk, Vignesh Gunasekharan, Jun Zhao, Rihao Qu, Xiaotong Li, Gauri A. Patwardhan, Vikram B. Wali, Abhishek K. Gupta, Manoj M. Pillai, Yuval Kluger, Christos Hatzis, Lajos Pusztai. Genomic, transcriptomic, and epigenetic profiling of triple-negative breast cancer cells after Navitoclax treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6333.

Mutational landscape of CLL is now known to include recurrent non-synonymous mutations in SF3B1, a core splicing factor. About 5-10% of newly diagnosed CLL harbor these mutations which are typically limited to HEAT domains in the carboxyl-terminus of the protein. Importantly, the mutations are not specific to CLL but also present in several unrelated clonal disorders. Analysis of patient samples and cell lines has shown the primary splicing aberration in SF3B1-mutant cells to the use of novel or "cryptic" 3' splice sites (3SS). Advances in genome-editing and next-generation sequencing (NGS) have allowed development of isogenic models and detailed analysis of changes to the transcriptome with relative ease. In this manuscript, we focus on two relevant methods to study splicing factor mutations in CLL: development of isogenic scalable cell lines and informatics analysis of RNA-Seq datasets.

Mutational landscape of CLL is now known to include recurrent non-synonymous mutations in SF3B1, a core splicing factor. About 5-10% of newly diagnosed CLL harbor these mutations which are typically limited to HEAT domains in the carboxyl-terminus of the protein. Importantly, the mutations are not specific to CLL but also present in several unrelated clonal disorders. Analysis of patient samples and cell lines has shown the primary splicing aberration in SF3B1-mutant cells to the use of novel or "cryptic" 3' splice sites (3SS). Advances in genome-editing and next-generation sequencing (NGS) have allowed development of isogenic models and detailed analysis of changes to the transcriptome with relative ease. In this manuscript, we focus on two relevant methods to study splicing factor mutations in CLL: development of isogenic scalable cell lines and informatics analysis of RNA-Seq datasets.

Disruption of post-transcriptional gene regulation is a critical step in oncogenesis that can be difficult to observe using traditional molecular techniques. To overcome this limitation, a modified polyadenylation site sequencing (PAS-seq) protocol was used to generate a genome-wide map of alternative polyadenylation (APA) events in human primary breast tumor specimens and matched normal tissue. This approach identified an APA event in the PRELID1 mRNA that enhances its steady state level and translational efficiency, and is a strong breast cancer subtype-dependent predictor of patient clinical outcomes. Furthermore, it is demonstrated that PRELID1 regulates stress response and mitochondrial reactive oxygen species (ROS) production in a cell-type specific manner. Modulation of PRELID1 expression, including its post-transcriptional control, appears to be a common stress response across different cancer types. These data reveal that PRELID1 mRNA processing is an important regulator of cell-type specific responses to stress used by multiple cancers and is associated with patient outcomes.

E2F-2 is a retinoblastoma (Rb)-regulated transcription factor induced during terminal erythroid maturation. Cyclin E-mediated Rb hyperphosphorylation induces E2F transcriptional activator functions. We previously reported that deregulated cyclin E activity causes defective terminal maturation of nucleated erythroblasts in vivo. Here, we found that these defects are normalized by E2F-2 deletion; however, anemia in mice with deregulated cyclin E is not improved by E2F-2-loss, which itself causes reduced peripheral red blood cell (RBC) counts without altering relative abundances of erythroblast subpopulations. To determine how E2F-2 regulates RBC production, we comprehensively studied erythropoiesis using knockout mice and hematopoietic progenitors. We found that efficient stress erythropoiesis in vivo requires E2F-2, and we also identified an unappreciated role for E2F-2 in erythroblast enucleation. In particular, E2F-2 deletion impairs nuclear condensation, a morphological feature of maturing erythroblasts. Transcriptome profiling of E2F-2-null, mature erythroblasts demonstrated widespread changes in gene expression. Notably, we identified citron Rho-interacting kinase (CRIK), which has known functions in mitosis and cytokinesis, as induced in erythroblasts in an E2F-2-dependent manner, and we found that CRIK activity promotes efficient erythroblast enucleation and nuclear condensation. Together, our data reveal novel, lineage-specific functions for E2F-2 and suggest that some mitotic kinases have specialized roles supporting enucleation of maturing erythroblasts.

miRNAs regulate the expression of genes in both normal physiology and disease. While miRNAs have been demonstrated to play a pivotal role in aspects of cancer biology, these reports have generally focused on the regulation of single genes. Such single-gene approaches have significant limitations, relying on miRNA expression levels and heuristic predictions of mRNA-binding sites. This results in only circumstantial evidence of miRNA-target interaction and typically leads to large numbers of false positive predictions. Here, we used a genome-wide approach (high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation, HITS-CLIP) to define direct miRNA-mRNA interactions in three breast cancer subtypes (estrogen receptor positive, Her2 amplified, and triple negative). Focusing on steroid receptor signaling, we identified two novel regulators of the ER pathway (miR-9-5p and miR-193a/b-3p), which together target multiple genes involved in ER signaling. Moreover, this approach enabled the definition of miR-9-5p as a global regulator of steroid receptor signaling in breast cancer. We show that miRNA targets and networks defined by HITS-CLIP under physiologic conditions are predictive of patient outcomes and provide global insight into miRNA regulation in breast cancer.

Regulation of hematopoietic stem cell proliferation, lineage commitment, and differentiation in adult vertebrates requires extrinsic signals provided by cells in the marrow microenvironment (ME) located within the bone marrow. Both secreted and cell-surface bound factors critical to this regulation have been identified, yet control of their expression by cells within the ME has not been addressed. Herein we hypothesize that microRNAs (miRNAs) contribute to their controlled expression. MiRNAs are small noncoding RNAs that bind to target mRNAs and downregulate gene expression by either initiating mRNA degradation or preventing peptide translation. Testing the role of miRNAs in downregulating gene expression has been difficult since conventional techniques used to define miRNA-mRNA interactions are indirect and have high false-positive and negative rates. In this report, a genome-wide biochemical technique (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation or HITS-CLIP) was used to generate unbiased genome-wide maps of miRNA-mRNA interactions in two critical cellular components of the marrow ME: marrow stromal cells and bone marrow endothelial cells. Analysis of these datasets identified miRNAs as direct regulators of JAG1, WNT5A, MMP2, and VEGFA; four factors that are important to ME function. Our results show the feasibility and utility of unbiased genome-wide biochemical techniques in dissecting the role of miRNAs in regulation of complex tissues such as the marrow ME. Stem Cells 2014;32:662–673

Introduction: TP53 mutations have been associated with adverse outcomes in AML and MDS. While these mutations occur in less than 15-20% of pts, they are often associated with complex karyotypes and therapy-related (t)-myeloid neoplasms (MN). Previous studies showed poor outcomes with intensive chemotherapy (IC) and lower-intensity therapies [LIT] (e.g. hypomethylating agents [HMA]), and higher relapse rates after allogeneic hematopoietic stem cell transplant (alloSCT), leaving the question of optimal treatment unanswered. Methods: We conducted a retrospective review of all adult pts with MN and pathogenic TP53 mutations who were treated at Yale University from 9/1/2015 to 5/31/2019. We collected data on age, sex, ethnicity, prior malignancies and their treatments, white blood cell (WBC), hemoglobin, platelet count, disease risk, initial and subsequent lines of therapies, and use of alloSCT. Responses were recorded using modified International Working Group (IWG) criteria 2003 for AML and 2006 for MDS. Overall survival (OS) was measured using Kaplan Meier methods. Results: We identified 83 pts with TP53-mutated MN (45 AML, 31 MDS, 4 chronic myelomonocytic leukemia, and 3 JAK2-positive myeloproliferative neoplasms). Median age was 69 years (range [R], 27-88 years), 52% were male, and 88% were Caucasian [Table 1]. Prior malignancy was noted in 35 pts (42%) and 29 (17 AML, 12 MDS) pts were classified as t-MN. Median WBC on presentation was 3 x 109 /L (R, 0.2 - 74x109) with a median of 0% peripheral blasts (R, 0-43%). Complex (n=75; 90%) and monosomal karyotypes (n=54; 65%) were highly prevalent. We identified 101 unique, pathogenic or likely pathogenic mutations in the TP53 gene with 17 pts harboring more than 1 abnormal variant (16 pts with 2 mutations, 1 pt with 4 mutations). Missense variants were most commonly encountered (n=82 variants, 81%) followed by splicesite (n=9, 9%), frameshift (n=6, 6%), and nonsense variants (n=4, 4.0%). 52 pts had isolated somatic TP53 variants (63%). Among the other 31 pts DNMT3A (n=8 pts), JAK2 (n=6), and TET2 (n=5) variants were the most common somatic mutations. Of note, targetable driver mutations such as FLT3 (n=0), IDH1 (n=3), and IDH2 (n=1) were very rare. Notably, pts with complex karyotype were more likely to have isolated TP53 mutations compared to non-complex karyotype pts (complex karyotype + isolated TP53 mutations: 51 pts [68.0%] vs non-complex karyotype + isolated TP53 mutations: 2 pts [25.0%]; Fisher's exact test: p=0.024) [Table 2]. Median follow up of the cohort was 6.4 months (mos) [R: 0.2-55.3 mos]. Median OS in the entire study cohort was 7.6 mos (95% CI: 5.7-10.0 mos) with a 1-year OS rate of 30.5% (95% CI: 20.0-41.6%). Response rates and outcome for AML and MDS patients by treatment regimen are shown in Table 3. Among AML pts, IC did not appear to improve OS compared to pts receiving LIT (median OS 10.1 mos [95% CI: 6.4-15.5] vs 8.1 mos [95% CI: 1.9-13.6]; p=0.39). AML pts (median OS 6.7 mos [95% CI: 1.9-9.4]; 1-year OS: 23.2% [95% CI: 11%-38%]) had a statistically non-significant trend to poorer outcomes compared to MDS pts (median OS 10 mos [95% CI 5.7-12.9]; 1-year OS: 29.5% [95% CI 13.5-47.4%]; p=0.13). Notably, among the 10 pts who proceeded to alloSCT, only 3 pts relapsed after a median of 2.8 mos post-alloSCT. Relapse-free survival and median OS for pts who underwent alloSCT was not reached at a median follow up of 10.4 mos. Conclusions: In this case series we confirm the poor prognosis for pts with TP53-mutated MN. Median OS was only 7.6 mos, and IC did not appear to improve OS compared to LIT for AML pts. With limited follow-up, pts who underwent alloSCT appeared to benefit. Novel therapies are urgently needed to improve outcomes of this pt population. Disclosures Podoltsev: Kartos Therapeutics: Research Funding; Samus Therapeutics: Research Funding; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Sunesis Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding; CTI Biopharma: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; AI Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding. Gore:Celgene Corporation: Consultancy, Research Funding. Prebet:Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; pfizer: Honoraria; pfizer: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; novartis: Honoraria; Agios: Consultancy, Research Funding; novartis: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; pfizer: Honoraria; Genentech: Consultancy; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; novartis: Honoraria; novartis: Honoraria. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:Spectrum: Other: fees for non-CME/CE services ; Eisai: Consultancy; Acrotech: Consultancy; miRagen: Consultancy; Mallinckrodt: Consultancy; Seattle Genetics: Consultancy, Other: fees for non-CME/CE services . Huntington:AbbVie: Consultancy; Celgene: Consultancy, Research Funding; DTRM Biopharm: Research Funding; Pharmacyclics: Honoraria; Genentech: Consultancy; Bayer: Consultancy, Honoraria. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Zeidan:Celgene Corporation: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Otsuka: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria; Acceleron Pharma: Consultancy, Honoraria, Research Funding.

Introduction: Isolated trisomy 11 is a rare, poorly characterized cytogenetic abnormality in myeloid neoplasms (MN) with only a few case series published. Prior reports suggested a lower response rate to standard treatments such as intensive chemotherapy (IC) and lower-intensity therapy (e.g. hypomethylating agents [HMA]), as well as higher relapse rates after allogeneic hematopoietic stem cell transplant (alloSCT) and poor overall survival (OS) [Table 1]. We sought to define the clinical and molecular characteristics of patients (pts) and their clinical outcomes. Methods: We conducted a single center retrospective review of all adult pts with MN who were treated at Yale University from 1/1/2009 to 5/31/2019 and had trisomy 11 as the only cytogenetic abnormality. We collected data on age, sex, ethnicity, prior malignancies and their treatments, white blood cell (WBC), hemoglobin, platelet count, disease risk, initial and subsequent lines of therapies, and use of alloSCT. Responses were recorded using modified International Working Group (IWG) criteria 2003 for AML. OS was measured using Kaplan Meier methods. Molecular mutations were studied using a standard inhouse 40-gene next generation sequencing panel. Results: We identified 11 pts with isolated trisomy 11 on initial presentation, of whom 10 had AML (de novo AML [8], AML with myelodysplasia-related changes [2]), while one had chronic myelomonocytic leukemia (CMML). Median age was 64 years (range [R] 43-78 years), 91% were male, and 91% were Caucasian (Table 2). There were no cases of therapy-related MN (t-MN) or myelodysplastic syndromes (MDS). Median WBC count on initial presentation was 19x109 /L (R: 1.2-232 x109) with a median of 42% peripheral blasts (R: 0-99%), and 2 pts presented with hyperleukocytosis (WBC >100 x109 /L). All cases of trisomy 11 on karyotype were also positive by fluorescence in-situ hybridization. Testing for somatic mutations was performed in 9 pts. Twenty somatic mutations were detected in 8 pts with the most common mutations affecting IDH2 (n=4 pts, 36.4%), IDH1 (n=3, 27.3%), DNMT3A (n=2, 18.2%), and U2AF1 (n=2; 18.2%). For initial therapy of the 10 AML pts, 6 pts were treated with intensive chemotherapy (IC) with 3 pts receiving 7+3 (one of whom previously received leukapheresis), one receiving high dose cytarabine, and 2 others receiving IC on clinical trials. Three pts were treated with HMA-based therapies, while the 10th pt received supportive care (SC) only. The CMML pt was treated on a clinical trial with an oral HMA. Among AML pts, 6 pts achieved complete remission [CR] (including 5 of 6 pts treated with IC, and one out of 3 pts who received HMA), while the other 4 were primary refractory. Three pts relapsed during study follow-up. Of the 7 pts with relapsed/refractory (R/R)-AML, gemtuzumab ozogamicin + decitabine, an anti-CD47 antibody on trial, low-dose cytarabine + venetoclax (LDAC+VEN), and IC with cytarabine + mitoxantrone were used for 4 pts while the other 3 pts received only SC. Only one pt with R/R AML responded (he received LDAC + VEN) with a CR with incomplete count recovery (CRi). Most of these pts were treated before the approval of IDH inhibitors and therefore only one pt received an IDH inhibitor in second relapse. Six pts died during the study period while 5 were still alive at last follow up. Median duration of follow up was 14 months (R: 1-53 months). Three pts underwent alloSCT, all during first CR. Median OS of the entire cohort was 30 months (95%CI: 1.8 months - not reached). After censoring at the time of alloSCT, median OS was 20 months (95% CI: 2 months-not reached, [Figure 1]). A durable CR after alloSCT was observed in 1 pt (alive and in CR at 51 months after alloSCT), while another pt relapsed 8 months after alloSCT. A third pt remains in CR 8 months after alloSCT. Interpretation: Here we report our single-center experience in MN pts with the very rare cytogenetic abnormality of trisomy 11. We confirm that this abnormality is extremely rare in MDS but can be seen in CMML. While in AML it is also rare, it does not seem to occur in the context of t-AML, and it tends to coexist with the targetable IDH-1/2 mutations. While we also confirm that survival is generally poor, OS among our pts appeared substantially longer than in prior studies with one pt being alive and in CR at 51 months after alloSCT. Disclosures Podoltsev: CTI Biopharma: Research Funding; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Sunesis Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Jazz Pharmaceuticals: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; AI Therapeutics: Research Funding; Samus Therapeutics: Research Funding; Arog Pharmaceuticals: Research Funding; Kartos Therapeutics: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding. Gore:Celgene Corporation: Consultancy, Research Funding. Prebet:pfizer: Honoraria; novartis: Honoraria; pfizer: Honoraria; Agios: Consultancy, Research Funding; pfizer: Honoraria; pfizer: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; pfizer: Honoraria; Boehringer Ingelheim: Research Funding; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; novartis: Honoraria; Tetraphase: Consultancy; Boehringer Ingelheim: Research Funding; novartis: Honoraria; novartis: Honoraria; Genentech: Consultancy; novartis: Honoraria; Boehringer Ingelheim: Research Funding. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:miRagen: Consultancy; Eisai: Consultancy; Seattle Genetics: Consultancy, Other: fees for non-CME/CE services ; Spectrum: Other: fees for non-CME/CE services ; Acrotech: Consultancy; Mallinckrodt: Consultancy. Huntington:Genentech: Consultancy; Pharmacyclics: Honoraria; Celgene: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; DTRM Biopharm: Research Funding; AbbVie: Consultancy. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Zeidan:Otsuka: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Seattle Genetics: Honoraria; BeyondSpring: Honoraria.

Among genetic aberrations responsible for ineffective hematopoiesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia, somatic mutations in splicing factors such as U2AF1 are of significant interest as they are recurrent, mutually exclusive and early occurring. U2AF1 participates in mRNA splicing through the recognition of the intronic 3' splice site, forming the U2AF complex as a heterodimer with U2AF2. Heterozygous hotspot mutations at S34 or Q157, in the two U2AF1 zinc fingers respectively, result in sequence dependent aberrant splicing, suggestive of altered RNA binding. The mechanism by which these mutations alter U2AF1-U2AF2-RNA interactions has to date not been elucidated, yet understanding the structure-function relationship is critical to devise novel therapeutic strategies that either aim to correct or exploit RNA binding and splicing defects. To address this issue, we profiled the transcriptome of HEL erythroleukemic cell lines expressing wild-type (WT) and mutant U2AF1. U2AF1 S34F and Q157R mutants induced widespread alterations in splicing patterns of 3250 and 1791 genes respectively, with an overlap of 23.8% genes. On the other hand, we observed only minor alterations in gene expression levels. Meta-analysis and comparison with published RNA sequencing datasets on U2AF1 mutants revealed both conserved and unique splicing changes, with a strong enrichment for genes involved in cell cycle (P=6.7E-15) and DNA repair (P=2.6E-5). Confirming previous literature, U2AF1 S34F preferentially leads to the exclusion of exons preceded by 3' splice sites bearing an intronic UAG motif, while U2AF1 Q157R preferentially excludes exons starting with the AGA motif (Figure 1A). Collectively, the S34F mutation has a stronger effect on splicing, ultimately decreasing the global translation state of cells. To understand how mutations eventually result in the observed splicing alterations, we also profiled with unprecedented resolution the RNA interactome of the physiological and pathological U2AF heterodimer. We first performed enhanced crosslinking immunoprecipitation (eCLIP) on U2AF1 WT, U2AF1 mutants and U2AF2. Comparison of U2AF1 and U2AF2 binding profiles revealed a high degree of similarity, suggesting that they mostly bind to RNA as a tight dimer. Only by performing fractionated eCLIP on U2AF1 we were able to isolate, at the molecular level, the individual contributions of the U2AF components in the recognition of the 3' splice site. In particular, we deconvolved the U2AF2 signal, insisting on the polypyrimidine region, and the U2AF1 signal, peaking on the AG dinucleotide at the intronic end (Figure 1B). Importantly, the S34F mutant displays an aberrant binding profile, with a specific peak on the nucleotide in position -3, matching the sequence specificity previously observed in aberrant splicing events (Figure 1A-B). Systematic analysis of bound junctions suggests a complex model where the S34F mutation does not simply abrogate the ability of U2AF1 to bind splicing junctions ending with the UAG sequence, but rather alters the conformation of the U2AF complex bound to RNA, resulting in a differential ability to effectively recruit the U2 complex. To confirm this model, we identified and validated a set of gain-of-function splice junctions in genes contributing to hemopoiesis and cell cycle, characterized by increased binding of U2AF1 S34F mutant and parallel decreased binding of U2AF2. In summary, we identified novel RNA sequence and structure determinants of U2AF complex conformation, uncovered by the binding alterations induced by the U2AF1 S34F mutation. Our data further dissect the complexities of post-transcriptional regulation and provide the basis for development of U2AF directed cancer therapies. Disclosures Hunck: B**hringer-Ingelheim Foundation.: Other: During my stay in the Halene Lab I was founded by an MD fellowship.

Introduction: While the use of immune checkpoint inhibitors (ICI) is now an established standard of care in the management of many solid tumors, the efficacy of this approach in myeloid neoplasms (MN) is still being explored. Preliminary data regarding efficacy presented to date are modest at best. Since many leukemia physicians lack extensive experience using ICI, and MN patients experience deep thrombocytopenia and bleeding risk precluding lung or colon biopsies to assess for immune-related adverse events (irAEs), MN presents a particularly difficult space for ICI development. Particular concern may center around distinguishing progressive infections (especially fungal) from immune pneumonitis and the appropriateness of steroids in such settings We report a single-center experience on the use of ICI in MN. Methods: We retrospectively analyzed data from patients (pts) with acute myeloid leukemia (AML) or higher-risk (International Prognostic Scoring System [IPSS] INT-2 or high-risk) myelodysplastic syndromes (MDS) treated with ICI therapy from April 16th, 2015 to June 3rd, 2019. We collected data on patient age, sex, race, Performance Status (ECOG PS), WBC, hemoglobin (Hgb), platelet count, serum metabolic and coagulation parameters, AML disease risk (cytogenetic and molecular) or MDS risk by IPSS score, prior lines of therapy (for refractory/relapsed [R/R] pts), ICI used (classified by targeted immune checkpoint to maintain trial confidentiality) as well as concurrent leukemia-directed therapy, irAEs, transfusion requirements, response to therapy and overall survival (OS). Results: Twenty-seven (13 AML and 14 MDS) pts received ICI in the context of clinical trials. Median age was 73 years, 63% were male, and 95% had an ECOG PS of 0 or 1 (Table 1). Nineteen pts (59%) had R/R disease, of whom 14 previously-received hypomethylating agent (HMA) therapy while 5 received intensive induction therapy. Median number of prior therapies was 1 (range [R], 1-3). Median follow-up was 11.3 months (R, 0.5-46.7). 54% of AML pts had cytogenetically or molecularly-defined poor risk disease; 86% of MDS pts had IPSS high-risk disease. Complex karyotype and TP53 mutation was noted in 35% and 35% of pts, respectively. (Table 1). Fifteen pts (56%) received a PDL1 inhibitor in combination with a HMA, while 4 (19%) pts received either a PD1 inhibitor in combination with a histone deacetylase inhibitor or CTLA4 inhibitor monotherapy. Two pts (7%) received dual ICI with both a PDL1 inhibitor and CTLA4 inhibitor (Table 1). The median number of ICI cycles administered was 4 with the main reason for discontinuation being lack of response (39%). Notably 27% discontinued because of intolerance to therapy. Almost half (n=12, 44%) of pts experienced an irAE; 4 pts had a grade 3 irAE. There were no grade 4 irAEs, but one patient with MDS developed multi-organ grade 3 irAEs and despite appropriate steroid therapy died due to irAEs with necrotizing myocarditis (with multifocal lymphocytic inflammatory infiltrate) and pericarditis confirmed on post-mortem examination. Of pts experiencing an irAE, 69% (including all pts with grade 3 irAEs) received high-dose corticosteroid therapy with a median time to initiation of 3 days (R, 1-14). No biopsies were performed due to bleeding risk in setting of cytopenia. Two of six newly-diagnosed AML pts achieved CR in response to ICI+HMA; one of 2 newly-diagnosed MDS pts developed CR following ICI+HMA. No R/R AML or MDS pts responded. Half of pts proceeded to an additional line of therapy after ICI. Two pts (7%) proceeded to allogeneic stem cell transplant (alloSCT), both receiving anti-CTLA4 monotherapy x 4 cycles with last cycle 62 and 400 days prior to alloSCT, respectively. One pt developed stage 1 acute graft versus host disease (GVHD) of gut with complete response to steroids and another with moderate, classic chronic GVHD without unusual GVHD. No AML pts in this study remain alive. Median OS for newly-diagnosed AML pts treated with ICI + HMA was longer than those with R/R disease (11.6 vs. 5.0 months) at a median follow-up of 11.6 months (Table 2). Conclusions: Patients with AML and MDS treated with ICI have a significant risk of irAEs requiring provider diligence and possibly quick and life-saving intervention. Larger prospective studies of ICI in AML and MDS patients are needed to better define the role of ICB in management of these cancers. Disclosures Podoltsev: Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AI Therapeutics: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; CTI Biopharma: Research Funding; Arog Pharmaceuticals: Research Funding; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Research Funding; Astellas Pharma: Research Funding; Daiichi Sankyo: Research Funding; Sunesis Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding; Pfizer: Research Funding; Astex Pharmaceuticals: Research Funding; Celgene: Other: Grant funding, Research Funding; Genentech: Research Funding; Samus Therapeutics: Research Funding; Kartos Therapeutics: Research Funding. Prebet:Genentech: Consultancy; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Research Funding; novartis: Honoraria; Boehringer Ingelheim: Research Funding; novartis: Honoraria; Tetraphase: Consultancy; novartis: Honoraria; pfizer: Honoraria; novartis: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Boehringer Ingelheim: Research Funding; pfizer: Honoraria; Agios: Consultancy, Research Funding; novartis: Honoraria; pfizer: Honoraria; pfizer: Honoraria. Gore:Celgene Corporation: Consultancy, Research Funding. Isufi:Celgene: Consultancy; Novartis: Consultancy; Astra Zeneca: Consultancy. Foss:Seattle Genetics: Consultancy, Other: fees for non-CME/CE services ; miRagen: Consultancy; Mallinckrodt: Consultancy; Spectrum: Other: fees for non-CME/CE services ; Acrotech: Consultancy; Eisai: Consultancy. Huntington:Celgene: Consultancy, Research Funding; Bayer: Consultancy, Honoraria; AbbVie: Consultancy; Pharmacyclics: Honoraria; Genentech: Consultancy; DTRM Biopharm: Research Funding. Neparidze:Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees; Janssen Scientific Affairs, LLC: Research Funding. Zeidan:Daiichi Sankyo: Honoraria; Cardinal Health: Honoraria; Otsuka: Consultancy, Honoraria, Research Funding; Seattle Genetics: Honoraria; Abbvie: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Medimmune/AstraZeneca: Research Funding; Boehringer-Ingelheim: Consultancy, Honoraria, Research Funding; Trovagene: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; ADC Therapeutics: Research Funding; Jazz: Honoraria; Ariad: Honoraria; Agios: Honoraria; Novartis: Honoraria; Astellas: Honoraria; BeyondSpring: Honoraria; Acceleron Pharma: Consultancy, Honoraria, Research Funding; Celgene Corporation: Consultancy, Honoraria, Research Funding. OffLabel Disclosure: Immune checkpoint inhibitor therapy is not approved for use in patients with acute myeloid leukemia or myelodysplastic syndrome.

SF3B1 mutations are the most frequently occurring splicing factor mutations in MDS and AML, however the misspliced genes that contribute the malignant state in SF3B mutant MDS or AML remains unclear. We determined that SF3B1 mutant cases of MDS express a longer, active isoform of interleukin 1 receptor associated kinase (IRAK4). IRAK4 is a serine/threonine kinase that is downstream of toll-like receptor (TLR) signaling and leads to activation of oncogenic signaling states, including NF-kB and MAPK. Examination of IRAK4 by RNA sequencing showed that normal cells predominantly express small IRAK4 isoforms resulting from exclusion of the part of exon 6. These isoforms are targeted for proteosomal degradation leading to diminished IRAK4 expression and activation in normal cells. In contrast, a large proportion of MDS/AML samples with SF3B1 mutation show increased expression of an IRAK4 isoform that retains full exon 6, encoding the full-length protein (IRAK4-Long). Consequently, we show that expression of mutant SF3B1-K700E in leukemic cells is associated with increased NF-kB activity, suggesting that mutations in SF3B1 instruct expression of IRAK4 RNA isoforms with maximal functional potential. Furthermore, SF3B1 mutant MDS and AML cells exhibited a block in hematopoietic differentiation in clonogenic assays. This differentiation block was ameliorated with pharmacologic inhibition of IRAK4 with CA-4948, a potent oral clinically useful small-molecule inhibitor of IRAK4. CA-4948 blocked TLR-stimulated cytokine release in various cell models and also led to decreased leukemic burden in mice xenografted with SF3B1 mutant MDS/AML cells. Finally, we determined that SF3B1 mutation induced IRAK4 activation led to TRAF6 mediated K63 ubiquitination of critical cell cycle and regulatory proteins directly implicated in oncogenesis. We had recently shown that U2AF1 mutations can lead to IRAK4 activation via retention of exon 4 (Smith et al, Nat Cell Bio, 2019). Our data now demonstrate that SF3B1 leads to overactivation of IRAK4 via retention of a different exon (exon 6), thus reinforcing that IRAK/TRAF6 activation is a common downstream oncogenic pathway in splicing factor mutated MDS/AML. Taken together, in this study, we find that mutations in SF3B1 induce expression of therapeutically targetable "active" IRAK4 isoforms and provide a genetic link between a spliceosome mutation and oncogenic innate immune signaling in MDS and AML. Disclosures Booher: Curis: Employment. Ramachandra:Aurigene: Employment. Samson:Curis: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy; Aileron Therapeutics: Consultancy, Research Funding. Starczynowski:Kurome Therapeutics: Consultancy. Verma:Janssen: Research Funding; BMS: Research Funding; Celgene: Honoraria; Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria.

Introduction T-cell lymphomas are heterogenous group of neoplasms associated with generally unfavorable outcomes. High dose chemotherapy and autologous stem cell transplant (SCT) consolidation is a standard approach in first remission while allogeneic SCT (alloSCT) is recommended in the relapsed/refractory setting. Data on outcomes after alloSCT are limited, particularly in the era of brentuximab. Methods We retrospectively analyzed data from patients (pts) with T-cell lymphomas who underwent alloSCT in our institution from 11/2010–6/2018. Patient and disease characteristics were summarized using median and range for continuous data, and frequency and percentage for categorical data. Kaplan-Meier analysis was used to estimate progression free survival (PFS) and overall survival (OS) from the day of receipt of transplant. Survival probabilities were expressed by 95% confidence limits (CI). Results Thirty-three pts received alloSCT. Median age at transplant was 57 years (range [R], 22-73). T-cell lymphoma types included: cutaneous T-cell lymphoma, n = 12; NK-T cell, n = 4; peripheral T-cell lymphoma subtypes, n = 9; angioimmunoblastic T lymphoma, n = 6; T-cell rich B-cell, n = 2. Median lines of therapy prior to alloSCT were 4 (R, 2-13), including six with prior autologous SCT. Sixteen pts were transplanted in CR1, five in CR2, and three in CR3. Pentostatin/TBI was the most common pretransplant conditioning regimen (n = 23) followed by haplo regimen of Flu/TBI/Cy (n = 4). Donor transplant sources were MRD (16), MUD (8); Haplo (4), MMUD (5). Median days to neutrophil and platelet engraftment were 12 (R, 10-19) and 13 (R, 10-30), respectively. Cumulative incidence of grade II-IV acute and chronic GVHD were 24.2% and 45.4%, respectively. One-year PFS and OS probabilities were 69.5% (95% CI, 54.7- 88.4) and 87.1% (95% CI, 54.7-88.4), respectively. With a median follow up of 53 months (R, 2-97), 25 (75%) are alive and 24 are disease free. Four died from transplant related mortality (TRM) and 3 from relapse. The median time to relapse was 3.9 months [R, 2.2-12.5]). The cumulative incidence of GVHD was higher among patients who were relapse-free (71% vs 22% in relapsed pts, p=0.02). Seven of the 9 who relapsed were treated with brentuximab vedotin; of these 4 responded, including two patients who had haplo transplants. Conclusions Results from our study suggest that alloSCT in T-cell lymphomas with reduced intensity regimens are associated with overall favorable outcomes. Treatment of post-transplant relapse with brentuximab is feasible and should be explored further in long-term prospective studies.

Introduction Blood management and infectious disease mitigation are essential for success with allogeneic stem cell transplant (alloSCT). We sought to investigate the safety profile of a proactive infection mitigation strategy with the use of pathogen-inactivated (PI) platelets (PLT) during the alloSCT peri-engraftment period in the era of restrictive blood transfusion. Methods We retrospectively analyzed data from patients undergoing alloSCT that received prophylactic red blood cell (RBC) and PLT transfusion triggered by a hemoglobin of 8 g/dL and PLT count of 15,000 PLT/mL. When available, PI PLT (non-irradiated) were used starting November 2016 until March 2018 (study timeframe). Statistical analysis was performed using SAS 9.4 (Cary, NC). Patients characteristics and clinical parameters were summarized using mean and standard deviation or median and range for continuous data, and frequency and percentage for categorical data. Kaplan-Meier analysis was used to estimate progression-free survival (PFS) and overall survival (OS) from day of transplant. A two-sided logrank test was used to compare across strata using p Results Sixty-eight patients received alloSCT. Median age at transplant was 55 years (range [R], 18-73). Peripheral blood stem cells were the graft source in all but one patient. Donor source was a matched related, 10/10 matched unrelated (MUD), 9/10 MUD and haploidentical donor in 30.9%, 44.1%, 2.9%, and 22.1% of cases, respectively. Myeloablative conditioning was used in 45.6% of patients. Indication for alloSCT was a myeloid or lymphoid disorder in 64.7% and 35.3% of cases. Median time to neutrophil and PLT engraftment was 12 (R, 10-35) and 12 days (R, 8-120), respectively. Pre-alloSCT RBC alloimmunization was present in 11.8% of patients. Cumulative incidence of grades II-IV acute and chronic graft-versus-host disease (GVHD) was 20.6% and 22.1%, respectively. PI PLT were used in 73.5 % patients. There were no cases of transfusion associated graft failure (GF) or GVHD. Bacteremia occurred in 10.0% and 22.1% of patients by 30 days and 100 days post-alloSCT, respectively, and were both associated with worse OS (both p Conclusions Restrictive prophylactic transfusion thresholds in alloSCT recipients is feasible. Limited PI PLT transfusion was not associated with transfusion-attributable GVHD, GF or infectious complications. Future prospective studies should determine the short term hemostatic efficacy and long term post-alloSCT outcomes with restrictive transfusion criteria in an era of expanding PI product use.

Micro RNAs (miRNAs) are a class of small non-coding RNAs with well-known regulatory roles in normal physiological processes and cancer. Conventionally their study has been based on defining single miRNA-mRNA target interactions using a combination of miRNA expression arrays and bioinformatic predictions of binding to the 3′ untranslated regions of genes, followed by miRNA over-expression in the relevant cell type. These approaches present circumstantial evidence for binding of a particular miRNA to its target but do not allow the study of global interactions nor provide direct evidence of binding. In order to study the genome wide impact of miRNA regulation in breast cancer we chose to use a recently described biochemical technique called Cross-Linked Immune-Precipitation of Argonaute (AGO) protein followed by high throughput sequencing (HITS-CLIP). Cross-linking of RNA to adjacent protein moieties by ultraviolet (UV) light allows for stringent isolation of the miRNA-mRNA-AGO complexes by immune precipitation (IP); miRNA-mRNA interaction within the RNA-induced silencing complex (RISC) is known to occur within the folds of AGO. The isolated miRNA-mRNA-AGO complexes are then analyzed with next generation sequencing (NGS) to determine the miRNA-mRNA interactome. We performed HITS-CLIP on three well characterized breast cancer cell lines that represent ER+, Her2+ and triple negative (TN) disease (MCF7, BT474 and MDA231). To determine the role of miRNAs in coordinating the response to the estrogen receptor axis, MCF7 and BT474 cells were analyzed with or without short term (24 hour) estrogen treatment. Analysis of sequencing data revealed several novel miRNA-mRNA interactions that target the ER pathway. For example, miR-9 directly regulates ER expression and miR-193a is involved in regulating expression of the ER co-activator NCOA3. We confirmed the biological relevance of these results using functional in vitro studies where manipulation of both miR-193a and miR-9 altered the responsiveness of breast cancer cells to tamoxifen. These results were further validated by quantitation of both transcript (RT-PCR) and protein (Western Blot) levels after transfection of miRNA precursors. Functional binding between these miRNAs and putative binding targets in the 3′ Untranslated Regions (3′ UTRs) were also validated by luciferase-based reporter assays. Finally, we performed global analysis of miRNAs and their targets (as predicted by the HITS-CLIP datasets across all cell lines); this predicted regulation of core cellular processes such as cell proliferation, DNA repair and metabolism as being targeted by highly abundant miRNAs such as miR-27a and miR-21. In addition, our datasets confirmed previous reports of miRNA regulation of ER pathway such as miR-221 regulation of ER itself and miR-34a regulation of Myc. In summary, genome-wide biochemical approaches like HITS-CLIP allow for defining novel and clinically relevant miRNA-based regulatory pathways of endocrine responsiveness and resistance in breast cancer. In addition, breast cancer subtype specific biological pathways targeted by individual miRNAs can be predicted by this approach. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD01-07.

Objective Macrophages are critical components of diverse microenvironments (ME) in adulthood, as well as during embryogenesis. Their role in development precludes the use of gene-targeting and knockout approaches for studying their function. Hence, we proposed to create a macrophage-specific inducible transgenic mouse where genes can be turned on or off at will. Materials and Methods A transgenic mouse in which the reverse tetracycline activator (rtTA-M2) is expressed under the hCD68 promoter for macrophage-specific gene induction was developed and crossed with a second transgenic reporter mouse strain in which the gene for green fluorescent protein (GFP) is under the control of tetracycline responsive element promoter. After doxycycline induction of the double transgenic animals (designated CD68-rtTA-tet-GFP), inducible expression of GFP was characterized by multicolor flow cytometric analysis of blood, marrow, and spleen cells and by demonstration of GFP expression in fresh-frozen sections in diverse tissues. Results In bone marrow, inducible GFP expression was not confined to, or inclusive of, all cells expressing the classical macrophage markers, such as F4/80. However, GFP-expressing cells in thioglycollate-elicited peritoneal macrophages were also positive for F4/80 and monocyte-macrophage–specific 2 antigen. Interestingly, flow analysis also indicated little overlap between the F4/80 and CSF-1R–positive populations. Fresh-frozen samples of tissues known to contain macrophages revealed GFP-expressing cells with variable morphologies. Conclusion Our results show that the hCD68 promoter directs gene expression in a macrophage population distinct from that defined by classical monocyte-macrophage markers or promoters. Whether this population is functionally distinct remains to be established.

Purpose: Antiendocrine therapy remains the most effective treatment for estrogen receptor–positive (ER+) breast cancer, but development of resistance is a major clinical complication. Effective targeting of mechanisms that control the loss of ER dependency in breast cancer remains elusive. We analyzed breast cancer–associated fibroblasts (CAF), the largest component of the tumor microenvironment, as a factor contributing to ER expression levels and antiendocrine resistance. Experimental Design: Tissues from patients with ER+ breast cancer were analyzed for the presence of CD146-positive (CD146pos) and CD146-negative (CD146neg) fibroblasts. ER-dependent proliferation and tamoxifen sensitivity were evaluated in ER+ tumor cells cocultured with CD146pos or CD146neg fibroblasts. RNA sequencing was used to develop a high-confidence gene signature that predicts for disease recurrence in tamoxifen-treated patients with ER+ breast cancer. Results: We demonstrate that ER+ breast cancers contain two CAF subtypes defined by CD146 expression. CD146neg CAFs suppress ER expression in ER+ breast cancer cells, decrease tumor cell sensitivity to estrogen, and increase tumor cell resistance to tamoxifen therapy. Conversely, the presence of CD146pos CAFs maintains ER expression in ER+ breast cancer cells and sustains estrogen-dependent proliferation and sensitivity to tamoxifen. Conditioned media from CD146pos CAFs with tamoxifen-resistant breast cancer cells are sufficient to restore tamoxifen sensitivity. Gene expression profiles of patient breast tumors with predominantly CD146neg CAFs correlate with inferior clinical response to tamoxifen and worse patient outcomes. Conclusions: Our data suggest that CAF composition contributes to treatment response and patient outcomes in ER+ breast cancer and should be considered a target for drug development. Clin Cancer Res; 23(7); 1710–21. ©2016 AACR.

The marrow microenvironment consists of several different interacting cell types, including hematopoietic-derived monocyte/macrophages and nonhematopoietic-derived stromal cells. Gene-expression profiles of stromal cells and monocytes cultured together differ from those of each population alone. Here, we report that CXCL7 gene expression, previously described as limited to the megakaryocyte lineage, is expressed by monocytes cocultured with stromal cells. CXCL7 gene expression was confirmed by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR), and secretion of protein was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot. At least 2 stromal-derived activities, one yet to be identified, were required for optimal expression of CXCL7 by monocytes. NAP-2, the shortest form of CXCL7 detected in the coculture media, was confirmed to decrease the size and number of CFU-Meg colonies. The propeptide LDGF, previously reported to be mitogenic for fibroblasts, was not secreted by stimulated monocytes. The re-combinant form of LDGF produced in a prokaryotic expression system did not have biologic activity in our hands. The monocytic source of CXCL7 was also detected by immunohistochemistry in normal bone marrow biopsies, indicating an in vivo function. We conclude that stromal-stimulated monocytes can serve as an additional source for CXCL7 peptides in the microenvironment and may contribute to the local regulation of megakaryocytopoiesis.

Musashi 2 (MSI2) is an RNA binding protein (RBP) that regulates asymmetric cell division and cell fate of several cell types including hematopoietic stem and progenitor cells. MSI2 was recently shown to be transcriptionally up-regulated in aggressive myeloid leukemia including chronic myelogenous leukemia in blast crisis (CML-BC) and acute myeloid leukemia (AML). Loss-of-function studies have confirmed an important role for MSI2 in the aggressive biology of these leukemia, but the precise molecular mechanisms remain undefined. MSI2 is thought to bind to the 3' untranslated region (3'UTR) of target mRNA in a sequence-specific fashion and inhibit their translation, but functional targets of MSI2 are not known. We implemented two transcriptome-wide techniques to define these targets: iCLIP (individual nucleotide-resolution cross-link and immunoprecipitation) and polysome profiling. We first defined the RNA interactome of MSI2 in a CML-BC cell line (K562) by iCLIP of FLAG-tagged MSI2. iCLIP relies on the ability of ultraviolet (UV) radiation to cross-link RBPs to interacting RNA allowing for stringent immunoprecipitation of RBP-RNA complexes and subsequent analysis by next generation sequencing. iCLIP analysis revealed direct binding of MSI2 to transcripts of 5036 genes, specifically enriched in the 3'UTR. These iCLIP peaks were also enriched in UAG, the minimal sequence motif of MSI2 previously defined through biochemical assays. Given that MSI2 is ubiquitously expressed, but has very specific cellular functions, we hypothesized that only a small fraction of the direct targets demonstrable by iCLIP are functionally relevant. To determine those target mRNA, we performed translatome profiling of K562 cells (MSI2 knock-down and control cells) with polysome profiling. Polyribosomes or polysomes are aggregates of ribosomes assembled on efficiently translated transcripts. Comparing change in abundance of polysome-associated transcripts (polysome profiling) allows determination of quantitative changes to translation. Using polysome profiling, we found that transcripts from only 413 genes underwent translational up-regulation (without a corresponding change in total transcript abundance) in response to MSI2 knock-down, of which 319 were deemed to be direct MSI2 targets from iCLIP analysis. Translational targets thus defined were highly enriched in biological pathways such as differentiation, cell-cycling, DNA damage response and apoptosis. Specific transcripts thus identified included transcription factors critical to myeloid and leukemia biology (cJUN, MYC and SP1) and cell-cycle regulators (CDK1, CDKN1B, RAD21 and RB1). Using expression profiles of 91 CML patient samples (reported in Radich JP et al PNAS 2006 Feb 21;103(8)) we tested if changes in gene-expression during disease progression (from chronic phase to blast crisis) reflect the change in translation of transcription factors as predicted by the translatome analysis. Accordingly, we found that transcripts down-regulated in CML-BC were highly enriched in targets of JUN and SP1, confirming a primary role for MSI2 targets in progression of CML. In summary, our novel approach that integrates two transcriptome-wide techniques show that while thousands of transcripts are directly bound by MSI2, only a small proportion of these undergo translational repression. Functional targets thus identified include transcription factors and cell-cycle regulators critical to progression of myeloid leukemia. Our results will form the basis of further studies to explore therapeutic targeting of MSI2 in myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

Splicing factor 3B1 (SF3B1) is a member of the U2 snRNP complex that is a key regulator of RNA splicing. RNA splicing begins with the recognition of splice sites (ss) at the 5' and 3' ends of introns and ends with the removal of introns and joining of exons flanking them. SF3B1 plays an important role in this process by facilitating the recognition of the 3'ss. SF3B1 is frequently mutated in numerous cancers as well as the myelodysplastic syndromes (MDS). Mutations within the HEAT domain of the protein potentially contribute to disease pathogenesis. In addition to influencing splicing by binding to pre-mRNA, SF3B1 has been shown to affect splicing of exons by associating with them directly on chromatin via histone/nucleosome interactions. However, it is not understood if or how SF3B1 association with chromatin is regulated. Given that N-terminal serine and threonine residues on SF3B1 are known substrates of cyclin E-Cdk2, which phosphorylates histone subunits and other chromatin associated proteins, we hypothesized that CDK2 activity regulates SF3B1-nucleosome interactions. Although SF3B1 is phosphorylated during splicing catalysis, the function of this phosphorylation has remained unknown. We have now discovered, using nucleosome preparations and histone subunit co-immunoprecipitation assays in synchronized cells, that endogenous SF3B1 interacts with nucleosomes in a highly cell-cycle dependent manner, while total cellular abundance of SF3B1 remains invariant during cell cycle progression. In human and mouse cells, including hematopoietic cell lines, SF3B1 is excluded from chromatin during both G0 (quiescence) and G2/M phases of cell cycle. Notably, SF3B1 is enriched within chromatin maximally during S-phase. Unexpectedly, we found that the inhibition of Cdc2 (Cdk1) during G2/M enforces the SF3B1-chromatin interaction, pointing to a direct role for Cdc2 in restraining this interaction during mitosis. Further, SF3B1 loading onto chromatin during early cell cycle progression from G0 to S-phase is inhibited by Cdk2 inhibition. Thus, Cdk2 and Cdc2 appear to have antagonistic roles in controlling SF3B1-chromatin interactions during the cell cycle. Our findings suggest that Cdk activity may regulate the recruitment of the spliceosome machinery in order to coordinate splicing of particular transcripts with cell cycle progression. Current studies are focusing on how disease-associated mutations in the HEAT domain of SF3B1 affect the dynamics of its cell cycle-dependent interaction with nucleosomes and corresponding alterations to splicing outcomes. Disclosures No relevant conflicts of interest to declare.

Marrow Stromal Cells (MSCs) are relatively rare cells difficult to visualize in marrow biopsies or detect in aspirated marrow. Under specific conditions MSC can be expanded in vitro and the population can give rise to several mesenchymal lineages. “MSC” also refers to mesenchymal stem cells which implies that all cells in the population are multipotent. It is generally agreed that while there may be a few multipotent stem cells in an MSC population the majority are not stem cells. In either case MSC do not produce hematopoietic cells. Although MSCs have been isolated and characterized from several tissues, bone marrow is their most common source for research and clinical use. Primary MSC populations can be derived from bone marrow mononuclear cells with relative ease, but it is important to recognize the cellular heterogeneity within a culture and how this may vary from donor to donor. In this chapter, we will describe methodology to derive primary MSCs from bone marrow screens, an otherwise discarded byproduct of bone marrow harvests used for clinical transplantation. We will also describe some useful techniques to characterize and manipulate MSCs – both primary and immortalized cell lines.