Isoform-specific Knockdown Research Articles

Regulation of alternative polyadenylation isoforms of Timp2 is an effector event of RAS signaling in cell transformation.

Alternative polyadenylation (APA) generates mRNA isoforms with different lengths of the 3' untranslated region (3' UTR). The tissue inhibitor of metalloproteinase 2 (TIMP2) plays a key role in extracellular matrix remodeling under various developmental and disease conditions. Both human and mouse genes encoding TIMP2 contain two highly conserved 3'UTR APA sites, leading to mRNA isoforms that differ substantially in 3'UTR size. APA of Timp2 is one of the most significantly regulated events in multiple cell differentiation lineages. Here we show that Timp2 APA is highly regulated in transformation of NIH3T3 cells by the oncogene HRAS G12V . Perturbations of isoform expression with long 3'UTR isoform-specific knockdown or genomic removal of the alternative UTR (aUTR) region indicate that the long 3'UTR isoform contributes to the secreted Timp2 protein much more than the short 3'UTR isoform. The short and long 3'UTR isoforms differ in subcellular localization to endoplasmic reticulum (ER). Strikingly, Timp2 aUTR enhances secreted protein expression but no effect on intracellular proteins in reporter assays. Furthermore, downregulation of Timp2 long isoform mitigates gene expression changes elicited by HRAS G12V . Together, our data indicate that regulation of Timp2 protein expression through APA isoform changes is an integral part of RAS-mediated cell transformation and 3'UTR isoforms of Timp2 can have distinct impacts on expression of secreted vs. intracellular proteins.

463. ISOFORM SWITCHING EVENTS IN ESOPHAGEAL ADENOCARCINOMA MAY INFORM NEW THERAPEUTIC TARGETS THROUGH IDENTIFYING MOLECULAR MECHANISMS CONTRIBUTING TO POOR PATIENT OUTCOMES

Abstract Background Esophageal adenocarcinoma (EAC) represents a growing health problem characterized by rising incidence and poor prognosis due to late-stage diagnosis coupled with therapeutic resistance. Moreover, EAC is a cancer with a high mutational burden but lacks highly prevalent oncogenic drivers that can be successfully targeted. Herein, we investigated whether newly identified isoform switching events, defined as differential usage of gene transcripts between conditions, may reveal new therapeutic targets and inform the molecular mechanisms contributing to EAC resistance. Methods We conducted RNA-sequencing followed by isoform switching analysis using IsoformSwitchAnalyzeR on Barrett’s low-grade dysplasia (BE.LGD) compared to Barrett’s with high-grade dysplasia (BE.HGD)+EAC tissues alone or in combination with TP53 mutation. Patients were stratified into tertiles based on isoform fraction levels, followed by overall and cancer-free survival analysis using the Cox proportional-hazards model. To assess whether mortality-linked isoforms influence cancer cell growth, viability, migration, and response to chemotherapy (Paclitaxel and Carboplatin). Isoform-specific siRNA knockdown experiments targeting HM13 were performed in OE19 and OE33 EAC cell lines, with STRING protein prediction interaction analysis and western blots conducted to assess pathway and protein alterations. Results Twenty-one isoform switched genes were significantly linked with all-cause mortality in BE.LGD versus BE.HGD+EAC and similarly 20 were significantly associated with cancer-specific patient mortality with 14 shared between the two groups. With inclusion of TP53 mutation status, 13 isoform switched genes were significantly linked with cancer-specific mortality. HM13, DANT2, CFDP1, KRAS, SIMM6 were among the most significantly altered. Knockdown of the pro-cancer isoform of HM13 significantly inhibited EAC cell migration and induced EAC cell death and knockdown of HM13 combined with chemotherapeutic treatment exhibited synergistic effects in EAC cell lines. Protein interaction analysis supported that HM13 is linked to ER-stress associated degradation, cellular response to stress, and ER unfolded protein response which were validated using a panel of relevant proteins (i.e., PERK, IRE-1a, ATF4, BIP, P-JNK). Conclusions Our research identified isoform switching events significantly linked to all-cause mortality as well as cancer specific mortality among EAC patients with and without TP53 mutations. Knockdown of the pro-cancer isoform of HM13 isoform significantly inhibited EAC cell migration and induced EAC cell death with synergistic effects observed with chemotherapeutic agent treatment supporting a role for specific isoform switches in therapeutic sensitization and in this case via the ERAD and ER unfolded protein response pathways. Modulation of specific proteins with established roles in ERAD and ER unfolded responses following isoform specific HM13 knockdown lends further support to an important role for this isoform, especially in TP53 mutant EAC. In conclusion, isoform switches may inform molecular mechanisms contributing to poor patient outcomes and reveal new potential therapeutic targets for EAC.

AKT2 Loss Impairs BRAF-Mutant Melanoma Metastasis.

Despite recent advances in treatment, melanoma remains the deadliest form of skin cancer due to its highly metastatic nature. Melanomas harboring oncogenic BRAFV600E mutations combined with PTEN loss exhibit unrestrained PI3K/AKT signaling and increased invasiveness. However, the contribution of different AKT isoforms to melanoma initiation, progression, and metastasis has not been comprehensively explored, and questions remain about whether individual isoforms play distinct or redundant roles in each step. We investigate the contribution of individual AKT isoforms to melanoma initiation using a novel mouse model of AKT isoform-specific loss in a murine melanoma model, and we investigate tumor progression, maintenance, and metastasis among a panel of human metastatic melanoma cell lines using AKT isoform-specific knockdown studies. We elucidate that AKT2 is dispensable for primary tumor formation but promotes migration and invasion in vitro and metastatic seeding in vivo, whereas AKT1 is uniquely important for melanoma initiation and cell proliferation. We propose a mechanism whereby the inhibition of AKT2 impairs glycolysis and reduces an EMT-related gene expression signature in PTEN-null BRAF-mutant human melanoma cells to limit metastatic spread. Our data suggest that the elucidation of AKT2-specific functions in metastasis might inform therapeutic strategies to improve treatment options for melanoma patients.

Endogenous SOLOIST/MRTFB i4, a Neuronal Isoform of MKL2/MRTFB, Positively and Negatively Regulates SRF Target Immediate Early Genes in Neuro-2a Cells.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB) is a serum response factor (SRF) cofactor that is enriched in the brain and controls SRF target genes and neuronal morphology. There are at least four isoforms of MKL2/MRTFB. Among these, MKL2/MRTFB isoform 1 and spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4) are highly expressed in neurons. Although, when overexpressed in neurons, isoform 1 and SOLOIST/MRTFB i4 have opposing effects on dendritic morphology and differentially regulate SRF target genes, it is unknown how endogenous SOLOIST/MRTFB i4 regulates gene expression. Using isoform-specific knockdown, we investigated the role of endogenous SOLOST/MRTFB i4 in regulating the expression of other MKL2/MRTFB isoforms and SRF-target genes in Neuro-2a cells. Knockdown of SOLOIST/MRTFB i4 downregulated SOLOIST/MRTFB i4, while it upregulated isoform 1 without affecting isoform 3. Knockdown of SOLOIST/MRTFB i4 downregulated the SRF target immediate early genes egr1 and Arc, while it upregulated c-fos. Double knockdown of isoform 1 and SOLOIST/MRTFB i4 inhibited c-fos expression. Taken together, our findings in Neuro-2a cells suggest that endogenous SOLOIST/MRTFB i4 positively regulates egr1 and Arc expression. In addition, endogenous SOLOIST/MRTFB i4 may negatively regulate c-fos expression, possibly by downregulating isoform 1 in Neuro-2a cells.

DNAJB6 isoform specific knockdown: Therapeutic potential for limb girdle muscular dystrophy D1

Dominant missense mutations in DNAJB6, a co-chaperone of HSP70, cause limb girdle muscular dystrophy (LGMD) D1. No treatments are currently available. Two isoforms exist, DNAJB6a and DNAJB6b, each with distinct localizations in muscle. Mutations reside in both isoforms, yet evidence suggests that DNAJB6b is primarily responsible for disease pathogenesis. Knockdown treatment strategies involving both isoforms carry risk, as DNAJB6 knockout is embryonic lethal. We therefore developed an isoform-specific knockdown approach using morpholinos. Selective reduction of each isoform was achieved invitro in primary mouse myotubes and human LGMDD1 myoblasts, as well as invivo in mouse skeletal muscle. To assess isoform specific knockdown in LGMDD1, we created primary myotube cultures from a knockin LGMDD1 mouse model. Using mass spectrometry, we identified an LGMDD1 protein signature related to protein homeostasis and myofibril structure. Selective reduction of DNAJB6b levels in LGMDD1 myotubes corrected much of the proteomic disease signature toward wild type levels. Additional invivo functional data is required to determine if selective reduction of DNAJB6b is a viable therapeutic target for LGMDD1.

Isoform-specific knockdown of long and intermediate prolactin receptors interferes with evolution of B-cell neoplasms

Prolactin (PRL) is elevated in B-cell-mediated lymphoproliferative diseases and promotes B-cell survival. Whether PRL or PRL receptors drive the evolution of B-cell malignancies is unknown. We measure changes in B cells after knocking down the pro-proliferative, anti-apoptotic long isoform of the PRL receptor (LFPRLR) in vivo in systemic lupus erythematosus (SLE)- and B-cell lymphoma-prone mouse models, and the long plus intermediate isoforms (LF/IFPRLR) in human B-cell malignancies. To knockdown LF/IFPRLRs without suppressing expression of the counteractive short PRLR isoforms (SFPRLRs), we employ splice-modulating DNA oligomers. In SLE-prone mice, LFPRLR knockdown reduces numbers and proliferation of pathogenic B-cell subsets and lowers the risk of B-cell transformation by downregulating expression of activation-induced cytidine deaminase. LFPRLR knockdown in lymphoma-prone mice reduces B-cell numbers and their expression of BCL2 and TCL1. In overt human B-cell malignancies, LF/IFPRLR knockdown reduces B-cell viability and their MYC and BCL2 expression. Unlike normal B cells, human B-cell malignancies secrete autocrine PRL and often express no SFPRLRs. Neutralization of secreted PRL reduces the viability of B-cell malignancies. Knockdown of LF/IFPRLR reduces the growth of human B-cell malignancies in vitro and in vivo. Thus, LF/IFPRLR knockdown is a highly specific approach to block the evolution of B-cell neoplasms.

Isoform specific knockdown of the ETS transcription factor Pointed in Drosophila S2 cells.

Alternate splicing of the pointed ( pnt ) gene locus produces two major isoforms, PntP1 and PntP2. Understanding their individual contributions to key developmental processes and identification of their genome-wide transcriptional targets has been hampered by a number of factors including their essential roles during embryonic development, and co-expression in several tissues. siRNAs were designed to target isoform-specific exons that code for the unique N-terminal region of either PntP1 or PntP2. The efficacy and specificity of the siRNAs were examined by co-transfection of isoform specific siRNAs with plasmids encoding epitope tagged PntP1 or PntP2 in Drosophila S2 cells. All P1-specific siRNAs were demonstrated to knockdown PntP1 protein level to greater than 95%, while having nominal impact on PntP2 level. Similarly, PntP2 siRNAs while ineffective at eliminating PntP1, were shown to reduce PntP2 protein level by 87-99%.

459 Regulation of the keratinocyte progenitor to differentiation switch by alternative mRNA splicing

Alternative splicing (AS) of messenger RNAs diversifies gene function by generating protein isoforms with different biological properties. Over 95% of human genes are alternatively spliced, but the functional consequences of most AS events are still not known. Here, using long-read RNA sequencing, we identified 5,826 AS events associated with human epidermal differentiation (percent spliced index >10%, FDR <0.01). AS events were enriched in genes associated with histone modification and phosphorylation (p < 0.001). The most common type of AS was cassette exon splicing, in which an intervening exon between two other exons is included or skipped in the mRNA sequence. We functionally characterized a cassette exon in mitogen-activated protein kinase (MAP3K7), which had not been previously studied in human epidermis. Basal epidermal progenitors preferentially express a shorter MAP3K7 isoform but switch to the longer isoform in suprabasal, differentiated epidermis with inclusion of an additional 81 bp exon. Using isoform-specific knockdown and overexpression, we demonstrate that inclusion of a single exon in MAP3K7 is necessary and sufficient to transition human epidermal keratinocytes from a replicating progenitor state with high clonogenic capacity into a post-mitotic differentiated state. We also performed binding motif analysis of sequences adjacent to epidermal AS events which identified 26 RNA binding proteins predicted to mediate mRNA splicing in epidermal keratinocytes, including ten with previously unstudied roles in the skin. In summary, our study defines the repertoire of alternative mRNA splicing events in the human epidermis and demonstrates the potential of this resource to identify functional AS events and splicing RBPs that regulate epidermal differentiation.

Abstract 986: RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness

Abstract NGS studies implicate dysregulation of the splicing machinery in the development of diverse cancers. The X-chromosome RBM10 gene encodes an RNA-binding protein that modulates transcriptome-wide cassette exon splicing. Truncation and missense RBM10 mutations are enriched (11%) in non-anaplastic thyroid cancers of patients who died of metastatic disease. MSK-MET, an integrated pan-cancer cohort of tumor genomic and clinical outcome data showed RBM10 alterations associate with metastatic burden in thyroid cancer. Within the MSK-IMPACT thyroid cancer cohort, 44% of RBM10 alterations co-occur with RAS mutations. We developed a murine Rbm10 floxed allele, which results in a non-functional transcript. Thyroid-specific Rbm10 inactivation through Tpo-Cre did not induce a phenotype. When crossed with FR-HrasG12V mice, which upon recombination generate endogenous expression of HrasG12V, Hras/Rbm10 mice developed thyroid cancers, ~ 20% of which were metastatic to lung. Cell lines derived from these tumors showed high penetrance of lung metastases after tail vein or orthotopic implantation into the thyroid. We identified splicing targets of RBM10 by high depth RNAseq of 5 isogenic human thyroid cancer cell lines (2 RBM10-null with dox-induced RBM10; 3 RBM10 WT with RBM10 shRNA). The common abnormalities associated with RBM10 loss were exon inclusion events. Ingenuity Pathway Analysis of global transcriptomes in RBM10 isogenic human thyroid cancer cell lines showed enrichment in pro-migratory, aberrant integrin and RHO/RAC signaling expression signatures. Enriched GO analysis confined to genes subject to aberrant splicing by RBM10 loss extended these findings, with the top terms being cell adhesion, cytoskeleton, cadherin and integrin binding. RBM10 loss was associated with increased cell migration velocity in vitro as measured by time lapse imaging. Key cytoskeletal and extracellular matrix genes subject to exon inclusion events included vinculin (VCL), tenascin C (TNC), CD44, fibronectin (FN1) and tropomyosin 1 (TPM1). Isoform-specific knockdown of the VCL splice inclusion cassette exon that leads to illegitimate expression of metavinculin (mVCL) reduced cell migration, whereas isoform-specific knockdown of TNC and CD44 exon inclusion events reduced cell invasiveness in vitro. Rho GTPases transduce signals from the ECM to integrin receptors to regulate cell adhesion, migration, and invasiveness. Consistent with this, RBM10 overexpression in RBM10 null cells reduced RAC1-GTP levels. Finally, RBM10 re-expression in RBM10 null cells reversed metastatic competency in vivo. In conclusion, RBM10 loss alters the ratio of cassette exon inclusion events in a subset of transcripts that regulate interactions between the ECM and the cytoskeleton, leading to RHO/RAC activation and governing a process favoring increased cell movement and metastatic competence. Citation Format: Gnana P. Krishnamoorthy, Anthony Glover, Brian Untch, Mahesh Saqcena, Dina Vukel, Katherine Berman, Omar Abdel-Wahab, Robert K. Bradley, Jeffrey A. Knauf, James A. Fagin. RBM10 loss in thyroid cancer leads to aberrant splicing of cytoskeletal and extracellular matrix mRNAs and increased metastatic fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 986.

Investigation of the function of the PI3-Kinase / AKT signaling pathway for leukemogenesis and therapy of acute childhood lymphoblastic leukemia (ALL)

Acute lymphoblastic leukemia is the most common cause of cancer-related death in children and, especially for patients in a high-risk group, still represents a poor prognosis. The PI3K/AKT/mTOR signaling pathway has been identified as a frequently constitutively activated switching point in the disease of ALL. Despite the knowledge of the therapeutic importance of the signaling pathway, the results of clinically effective treatment strategies have so far been extremely sobering. In particular, monotherapy approaches represent a major problem with regard to cell resistance. In this work, the PI3K/AKT/mTOR signaling pathway was examined as a therapeutic target for the treatment of childhood acute lymphoblastic leukemia (ALL) with a new therapeutic approach to avoid cell resistance. Therefore, we used a combined therapeutic approach with inhibitors directed against AKT (MK2206), mTOR (RAD001) and the most prominent and aberrantly activated tyrosine kinase. In case of BCR-ABL-positive B-ALL cells we used a combination with the classic inhibitor Imatinib and in case of MLL-AF4-positive B-ALL cells we used a combination with Quizartinib (directed against FLT3). We show, in particular compared to the monotherapies, a highly significant inhibition of the growth of these cells after this new specific triple combination therapy. Furthermore, we show that inhibiting AKT alone leads to a feedback mechanism and an upregulation of the phosphorylation of a number of receptor-tyrosine-kinases. After isoform-specific knockdown of the three AKT isoforms in ALL cells we identified that especially ErbB2/Her2 is most strongly phosphorylated in cells with AKT2 knockdown. AKT isoform 1 and 2 knockdown cells show, in contrast to AKT isoform 3 knockdown cells, a weak proliferation and are presumably kept alive among others by the increased phosphorylation of the receptor-tyrosine-kinase ErbB2. This work provides first indications for a new combination therapy of B-ALL cells, which is directed against AKT, mTOR and a predominantly highly activated kinase.

LGMD: EP.176 DNAJB6 isoform specific knockdown: mechanistic insights and therapeutic potential for LGMD-D1

LGMD: EP.176 DNAJB6 isoform specific knockdown: mechanistic insights and therapeutic potential for LGMD-D1

Knockdown of AKT3 Activates HER2 and DDR Kinases in Bone-Seeking Breast Cancer Cells, Promotes Metastasis In Vivo and Attenuates the TGFβ/CTGF Axis.

Bone metastases frequently occur in breast cancer patients and lack appropriate treatment options. Hence, understanding the molecular mechanisms involved in the multistep process of breast cancer bone metastasis and tumor-induced osteolysis is of paramount interest. The serine/threonine kinase AKT plays a crucial role in breast cancer bone metastasis but the effect of individual AKT isoforms remains unclear. Therefore, AKT isoform-specific knockdowns were generated on the bone-seeking MDA-MB-231 BO subline and the effect on proliferation, migration, invasion, and chemotaxis was analyzed by live-cell imaging. Kinome profiling and Western blot analysis of the TGFβ/CTGF axis were conducted and metastasis was evaluated by intracardiac inoculation of tumor cells into NOD scid gamma (NSG) mice. MDA-MB-231 BO cells exhibited an elevated AKT3 kinase activity in vitro and responded to combined treatment with AKT- and mTOR-inhibitors. Knockdown of AKT3 significantly increased migration, invasion, and chemotaxis in vitro and metastasis to bone but did not significantly enhance osteolysis. Furthermore, knockdown of AKT3 increased the activity and phosphorylation of pro-metastatic HER2 and DDR1/2 but lowered protein levels of CTGF after TGFβ-stimulation, an axis involved in tumor-induced osteolysis. We demonstrated that AKT3 plays a crucial role in bone-seeking breast cancer cells by promoting metastatic potential without facilitating tumor-induced osteolysis.

RET isoforms contribute differentially to invasive processes in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging disease with poor survival rates, owing to late diagnosis and early dissemination. These tumors frequently undergo perineural invasion, spreading along nerves regionally and to distant sites. The RET receptor tyrosine kinase is implicated in increased aggressiveness, local invasion, and metastasis in multiple cancers, including PDAC. RET mediates directional motility and invasion towards sources of its neurotrophic factor ligands, suggesting that it may enhance perineural invasion of tumor cells towards nerves. RET is expressed as two main isoforms, RET9 and RET51, which differ in their protein interactions and oncogenic potentials, however, the contributions of RET isoforms to neural invasion have not been investigated. In this study, we generated total RET and isoform-specific knockdown PDAC cell lines and assessed the contributions of RET isoforms to PDAC invasive spread. Our data show that RET activity induces cell polarization and actin remodeling through activation of CDC42 and RHOA GTPases to promote directional motility in PDAC cells. Further, we show that RET interacts with the adaptor protein TKS5 to induce invadopodia formation, enhance matrix degradation and promote tumor cell invasion through a SRC and GRB2-dependent mechanism. Finally, we show that RET51 is the predominant isoform contributing to these RET-mediated invasive processes in PDAC. Together, our work suggests that RET expression in pancreatic cancers may enhance tumor aggressiveness by promoting perineural invasion, and that RET expression may be a valuable marker of invasiveness, and a potential therapeutic target in the treatment of these cancers.

Opposing Functions of BRD4 Isoforms in Breast Cancer

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.

Abstract P4-08-02: The human intermediate prolactin receptor is a breast cancer oncogene

Abstract Epidemiological, cellular, and genetic analyses indicate the hormone prolactin (PRL) and its cognate receptor in humans (hPRLr) are significantly involved in breast cancer pathogenesis. Recent evidence demonstrated that a truncated mouse PRLr (mPRLrT) is oncogenic when expressed alongside its canonical long form counterpart (mPRLrL). mPRLrT shares significant sequence homology with a naturally-occurring and widely-expressed hPRLr splice variant, the intermediate hPRLr (hPRLrI). To confirm the oncogenic potential of hPRLrI, isoform-specific hPRLrI knock-down (KD) was performed in breast cancer cell line MCF7. hPRLrI KD resulted in a significant decrease in proliferation, migration, and anchorage-independent growth. Next, given the similarity between mPRLrT and hPRLrI, we hypothesized hPRLrI may also be sufficient to induce transformation, when expressed alongside wild-type long hPRLr (hPRLrL). Like the mPRLrT, hPRLrI co-expression with hPRLrL in the immortalized but not transformed human breast cell line MCF10A resulted in a significant increase in proliferation, migration, and anchorage-independent growth. These results were not observed following overexpression of either isoform alone, demonstrating that hPRLrL/I co-expression is necessary to induce transformation of normal mammary epithelia. To test our hypothesis in vivo, we established MCF10A xenografts using female NOD scid gamma (NSG) mice. Following intraductal injection, we observed rapid tumor growth in the hPRLrL/I co-expression cohort, significantly over that of the cohorts harboring either isoform alone. To determine mechanisms of transformation, we examined both differential protein stability and altered signaling events. In analyzing receptor degradation, a cycloheximide assay revealed hPRLrL stability is increased when heterodimerized with hPRLrI. hPRLrL turnover has been reported to be impaired in human breast cancer, indicating this phenomenon may be involved in the observed hPRLrI-mediated transformation. In regards to differential signaling, we examined the Jak2/Stat5a pathway. Jak2 is a promiscuous kinase whose significant oncogenic actions are well-characterized, while Stat5a is a transcription factor whose activities are critical in attenuating the actions of Jak2. Following PRL stimulation, it was observed that hPRLrL/I co-expression induced approximately two-fold greater Jak2-Y1007/1008 phosphorylation (pJak2) compared to that induced by hPRLrL expression alone. Further, it was observed that hPRLrL/I co-expression induced ten-fold less Stat5a-Y694 phosphorylation (pY-Stat5a) than hPRLrL expression alone. These data indicate unchecked pJak2 activity may also be a contributing mechanism in the observed transformation. Overall, these results demonstrate that hPRLrI, alongside hPRLrL, is sufficient for transformation of normal breast tissue. Citation Format: Jacqueline M Grible, Shannon E Hedrick, Charles V Clevenger. The human intermediate prolactin receptor is a breast cancer oncogene [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-02.

The Splicing Factor RBM17 Supports Leukemia Stem Cell Self-Renewal

Background: Acute myeloid leukemia (AML) is thought to be sustained by sub-populations of leukemia stem cells (LSCs), which possess the capacity for self-renewal and differentiation and are believed to be responsible for disease initiation, relapse and chemoresistance. There is therefore an urgent need to develop therapies that target the LSC population to achieve effective AML treatment. To date there has been limited success in this endeavor, highlighting the importance of gaining a more comprehensive understanding of the mechanistic elements that underpin LSC function. Aberrant alternative splicing is recognized as a key driver of cancer. In the context of AML, genome-wide sequencing studies have shown that approximately one third of genes are differentially spliced in primitive CD34+ cells in AML patients compared to those obtained from normal controls. In particular, LSCs also have a unique splicing profile when compared to normal aging HSCs, underscoring the importance of understanding the mechanistic controls of aberrant splicing in LSC function. Results: Previous studies examining the link between aberrant splicing and AML focused on spliceosome genes with somatic mutations in AML patients. To examine mechanisms that mediate aberrant alternative splicing in LSC beyond splicing factor mutations, we performed a data-mining survey of 203 known mRNA splicing factors. Strikingly, RNA-binding motif protein 17 (RBM17) is the only splicing factor that is both strongly linked to poor AML prognosis and significantly elevated in LSC-enriched subsets of primary AML samples based on a recent study of 78 AML samples with normal karyotype. RBM17 has been implicated in regulating alternative splicing and cancer chemotherapy resistance. However, its function in AML or LSCs is not known. From our studies, we found that the level of RBM17 protein is elevated in the phenotypically primitive subsets of primary AML samples (n=8, RBM17+%: 67.42% in CD34+ versus 34.66% in CD34-fractions). Depletion of RBM17 with shRNAs in 3 human primary AML samples resulted in reduced colony formation compared to shScramble controls. More importantly, knockdown of RBM17 in a primary AML sample greatly impeded AML engraftment in immune-deficient mice (mean of 0.89 control versus mean of 0.1671 and 0.3171 shRNAs), suggesting that RBM17 is required for the stem and progenitor potential of AML and maintenance of LSC populations. Intriguingly, in contrast to the situation for the malignant hierarchy, the level of RBM17 in normal HSCs is lower than that in more committed cell populations in the normal hematopoietic system. To determine if RBM17 plays different roles in malignant LSC and normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBM17 with shRNAs in human cord blood (CB) derived CD34+ HSPCs, and found RBM17 knockdown had negligible adverse impact on both CB total colony and primitive GEMM colony outputs and yielded no increase in apoptosis, indicating no defects were apparent to primitive cells of CB as read out in vitro. To uncover the molecular mechanisms underlying the role of RBM17 in LSC functions, we preformed RBM17 eCLIP-seq in the K562 and HL60 human leukemic cell lines. We then cross-analyzed the CLIP-seq datasets with a published ENCODE RNA-seq dataset (RBM17 knockdown in K562 cells), where we found RBM17 directly binds to transcripts of stem cell program-related genes and regulates the splicing of these genes, including MADD (MAP kinase activating death domain) and MRPS18C (mitochondrial ribosomal protein S18C). We further demonstrated that the splicing patterns of MADD and MRPS18C are mediated by RBM17 in primary AML samples. In our ongoing functional validation experiments, isoform-specific knockdown of MADD or MRPS18C splice variants downstream of RBM17 impeded colony forming capacity and induced myeloid differentiation in the MOLM13 AML cell line. These results suggest that RBM17-mediated splicing events impact primitive cell function in AML. Conclusion: We have identified RBM17 as a novel LSC-regulating factor, plays an important role in maintaining AML LSC function through regulating the alternative splicing of stem cell program-related genes. The potential LSC-selective role for RBM17, along with its downstream splicing events, represent promising putative therapeutic targets whose modulation could offer attractive therapeutic windows in AML treatment. Disclosures No relevant conflicts of interest to declare.

Brain-specific Drp1 regulates postsynaptic endocytosis and dendrite formation independently of mitochondrial division.

Dynamin-related protein 1 (Drp1) divides mitochondria as a mechano-chemical GTPase. However, the function of Drp1 beyond mitochondrial division is largely unknown. Multiple Drp1 isoforms are produced through mRNA splicing. One such isoform, Drp1ABCD, contains all four alternative exons and is specifically expressed in the brain. Here, we studied the function of Drp1ABCD in mouse neurons in both culture and animal systems using isoform-specific knockdown by shRNA and isoform-specific knockout by CRISPR/Cas9. We found that the expression of Drp1ABCD is induced during postnatal brain development. Drp1ABCD is enriched in dendritic spines and regulates postsynaptic clathrin-mediated endocytosis by positioning the endocytic zone at the postsynaptic density, independently of mitochondrial division. Drp1ABCD loss promotes the formation of ectopic dendrites in neurons and enhanced sensorimotor gating behavior in mice. These data reveal that Drp1ABCD controls postsynaptic endocytosis, neuronal morphology and brain function.

Increased TGF-β and BMP Levels and Improved Chondrocyte-Specific Marker Expression In Vitro under Cartilage-Specific Physiological Osmolarity.

During standard expansion culture (i.e., plasma osmolarity, 280 mOsm) human articular chondrocytes dedifferentiate, making them inappropriate for autologous chondrocyte implantation to treat cartilage defects. Increasing the osmolarity of culture media to physiological osmolarity levels of cartilage (i.e., 380 mOsm), increases collagen type II (COL2A1) expression of human articular chondrocytes in vitro, but the underlying molecular mechanism is not fully understood. We hypothesized that TGF-β superfamily signaling may drive expression of COL2A1 under physiological osmolarity culture conditions. Human articular chondrocytes were cultured in cytokine-free medium of 280 or 380 mOsm with or without siRNA mediated TGF-β2 knockdown (RNAi). Expression of TGF-β isoforms, and collagen type II was evaluated by RT-qPCR and immunoblotting. TGF-β2 protein secretion was evaluated using ELISA and TGF-β bioactivity was determined using an established reporter assay. Involvement of BMP signaling was investigated by culturing human articular chondrocytes in the presence or absence of BMP inhibitor dorsomorphin and BMP bioactivity was determined using an established reporter assay. Physiological cartilage osmolarity (i.e., physosmolarity) most prominently increased TGF-β2 mRNA expression and protein secretion as well as TGF-β bioactivity. Upon TGF-β2 isoform-specific knockdown, gene expression of chondrocyte marker COL2A1 was induced. TGF-β2 RNAi under physosmolarity enhanced TGF-β bioactivity. BMP bioactivity increased upon physosmotic treatment, but was not related to TGF-β2 RNAi. In contrast, dorsomorphin inhibited COL2A1 mRNA expression in human articular chondrocytes independent of the osmotic condition. Our data suggest a role for TGF-β superfamily member signaling in physosmolarity-induced mRNA expression of collagen type II. As physosmotic conditions favor the expression of COL2A1 independent of our manipulations, contribution of other metabolic, post-transcriptional or epigenetic factors cannot be excluded in the underlying complex and interdependent regulation of marker gene expression. Dissecting these molecular mechanisms holds potential to further improve future cell-based chondral repair strategies.

VEGF165b, a splice variant of VEGF-A, promotes lung tumor progression and escape from anti-angiogenic therapies through a β1 integrin/VEGFR autocrine loop.

Vascular endothelial growth factor-A (VEGF-A) is highly subjected to alternative pre-mRNA splicing that generates several splice variants. The VEGFxxx and VEGFxxxb families encode splice variants of VEGF-A that differ only at the level of six amino acids in their C-terminal part. The expression level of VEGFxxx splice variants and their function as pro-angiogenic factors during tumor neo-angiogenesis have been well-described. The role of VEGFxxxb isoforms is less well known, but they have been shown to inhibit VEGFxxx-mediated angiogenesis, while being partial or weak activators of VEGFR receptors in endothelial cells. On the opposite, their role on tumor cells expressing VEGFRs at their surface remains largely unknown. In this study, we find elevated levels of VEGF165b, the main VEGFxxxb isoform, in 36% of non-small cell lung carcinoma (NSCLC), mainly lung adenocarcinoma (46%), and show that a high VEGF165b/VEGF165 ratio correlates with the presence of lymph node metastases. At the molecular level, we demonstrate that VEGF165b stimulates proliferation and invasiveness of two lung tumor cell lines through a VEGFR/β1 integrin loop. We further provide evidence that the isoform-specific knockdown of VEGF165b reduces tumor growth, demonstrating a tumor-promoting autocrine role for VEGF165b in lung cancer cells. Importantly, we show that bevacizumab, an anti-angiogenic compound used for the treatment of lung adenocarcinoma patients, increases the expression of VEGF165b and activates the invasive VEGFR/β1 integrin loop. Overall, these data highlight an unexpected role of the VEGF165b splice variant in the progression of lung tumors and their response to anti-angiogenic therapies.

Reversion to an Embryonic Alternative Splicing Program Enhances Leukemia Stem Cell Self-Renewal

BackgroundFormative research suggests that a human embryonic stem cell-specific alternative splicing gene regulatory network, which is repressed by Muscleblind-like (MBNL) RNA binding proteins, is involved in cell reprogramming. However, its role in malignant reprogramming of progenitors into self-renewing leukemia stem cells (LSCs) had not been established.MethodsWhole transcriptome RNA sequencing (RNA-seq) was performed on FACS purified progenitors from normal, chronic phase and blast crisis chronic myeloid leukemia samples and analyzed using Cuff-links, GSEA and IPA software. Splice isoform specific qRT-PCR, confocal microscopy, lentiviral overexpression and shRNA knockdown experiments were performed according to published methods (Jamieson NEJM 2004; Geron et al Cancer Cell 2008; Goff et al Cell Stem Cell 2013).ResultsWe performed LSC RNA-seq, lentiviral overexpression and knockdown and discovered that decreased expression of MBNL3, a repressor of an embryonic alternative splicing program and reprogramming, activated a pluripotency network and increased expression of a pro-survival isoform of CD44v3, which is more commonly expressed in human embryonic stem cells. This resulted in malignant reprogramming of progenitors in blast crisis CML endowing them with unbridled survival and self-renewal capacity. This is the first description of MBNL3 downregulation as a mechanism of reversion to an embryonic alternative splicing program, which elicits malignant progenitor reprogramming of progenitors into self-renewing leukemia stem cells. While isoform specific lentiviral CD44v3 overexpression enhanced chronic phase CML progenitor replating capacity, lentiviral shRNA knockdown abrogated these effects. In keeping with activation of a stem cell reprogramming network, CD44v3 upregulation was associated with increased expression of pluripotency transcription factors, including OCT4, SOX2 and b-catenin in addition to the pro-survival long isoforms of MCL1 and BCLX resulting in increased self-renewal and apoptosis resistance.ConclusionIn summary, MBNL3 downregulation activates an embryonic alternative splicing program, typified by CD44v3 overexpression, and represents a novel mechanism governing LSC generation in malignant microenvironments. Reversal of malignant reprogramming by epigenetic modulation of embryonic alternative splicing or via monoclonal antibody targeting of CD44v3 splice isoform may represent a pivotal opportunity for selective BC LSC eradication. DisclosuresJamieson:Johnson & Johnson: Research Funding; GlaxoSmithKline: Research Funding.