Khurshid Lab

Publications

Employing splice-switching oligonucleotides and AAVrh74.U7 snRNA to target insulin receptor splicing and cancer hallmarks in osteosarcoma

Molecular Therapy Oncology (2024)

Authors: Safiya Khurshid1,8 ∙ Akila S. Venkataramany1,2,3,8 ∙ Matias Montes4 ∙ John F. Kipp1 ∙ Ryan D. Roberts1,5 ∙ Nicolas Wein1 ∙ Frank Rigo6 ∙ Pin-Yi Wang1 ∙ Timothy P. Cripe1,5 ∙ Dawn S. Chandler1,7

Journal: Molecular Therapy Oncology (2024)

Abstract

Patients with osteosarcoma (OS), a debilitating pediatric bone malignancy, have limited treatment options to combat aggressive disease. OS thrives on insulin growth factor (IGF)-mediated signaling that can facilitate cell proliferation. Previous efforts to target IGF-1R signaling were mostly unsuccessful, likely due to compensatory signaling through alternative splicing of the insulin receptor (IR) to the proliferative IR-A isoform. Here, we leverage splice-switching oligonucleotides (SSOs) to mitigate IR splicing toward the IR-B isoform. We show that SSOs can modulate cancer cell hallmarks and anoikis-resistant growth. Furthermore, we engineered the SSO sequence in an U7 snRNA packaged in an adeno-associated virus (AAV) to test the feasibility of viral vector-mediated gene therapy delivery. We noted modest increases in IR-B isoform levels after virus transduction, which prompted us to investigate the role of combinatorial treatments with dalotuzumab, an anti-IGF-1R monoclonal antibody. After observing additive impacts on phosphoprotein phosphorylation and anoikis-resistant growth with the dalotuzumab and SSO combination, we treated OS cells with dalotuzumab and the AAVrh74.U7 snRNA IR virus, which significantly slowed OS cell proliferation. While these viruses require further optimization, we highlight the potential for SSO therapy and viral vector delivery, as it may offer new treatment avenues for OS patients and be translated to other cancers.

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Blockade of Interleukin-6 (IL-6) Signaling in Dedifferentiated Liposarcoma (DDLPS) Decreases Mouse Double Minute 2 (MDM2) Oncogenicity via Alternative Splicing

Bio Archives (2024)

Authors: View ORCID ProfileAbeba Zewdu, Danielle Braggio, Gonzalo Lopez, Kara Batte, Safiya Khurshid, Fernanda Costas de Faria, Hemant K. Bid, David Koller, Lucia Casadei, Katherine J. Ladner, David Wang, View ORCID ProfileValerie Grignol, O. Hans Iwenofu, Dawn Chandler, Denis C. Guttridge, View ORCID ProfileRaphael E. Pollock

Journal: Bio Archives (2024)

Abstract

Effective therapies for retroperitoneal (RP) dedifferentiated liposarcoma (DDLPS) remain unavailable. Loco-regional recurrence occurs in >80% of cases; 5-year disease-specific survival is only 20%. DDLPS is especially prevalent in the retroperitoneum and abdomen; evaluation of the DDLPS microenvironment in these high-fat compartments appears pertinent. Adipose is a main supplier of interleukin-6 (IL6); excessive activation of IL6 signal transducer glycoprotein 130 (GP130) underlies the development of some diseases. The role of GP130 pathway activation remains unstudied in DDLPS, so we examined the role of microenvironment fat cell activation of the IL6/GP130 signaling cascade in DDLPS. All DDLPS tumors and cell lines studied expressed elevated levels of the GP130-encoding gene IL6ST and GP130 protein compared to normal tissue and cell line controls. IL6 increased DDLPS cell growth and migration, possibly through increased signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) activation, and upregulated mouse double minute 2 (MDM2). GP130 loss conveyed opposite effects; pharmacological blockade of GP130 by SC144 produced the MDM2 splice variant MDM2-ALT1, known to inhibit full length MDM2 (MDM2-FL). Although genomic MDM2 amplification is pathognomonic for DDLPS, mechanisms driving MDM2 expression, regulation, and function beyond the MDM2:p53 negative feedback loop are poorly understood. Our findings suggest a novel preadipocyte DDLPS-promoting role due to IL6 release, via upregulation of DDLPS MDM2 expression. Pharmacological GP130 blockade reduced the IL6-induced increase in DDLPS MDM2 mRNA and protein levels, possibly through enhanced expression of MDM2-ALT1, a possibly targetable pathway with potential as future DDLPS patient therapy.

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Oncogenic Functions of Alternatively Spliced MDM2-ALT2 Isoform in Retroperitoneal Liposarcoma

MDPI (2024)

Authors: Fernanda Costas C. de Faria Fernanda Costas C. de Faria SciProfilesScilitPreprints.orgGoogle Scholar 1,Safiya Khurshid 2,Patricia Sarchet 1,Sayumi Tahara 1,Lucia Casadei 1,Valerie Grignol 1ORCID,Roma Karna 1,Sydney Rentsch 1,Nipin Sp 1,Joal D. Beane 1,Luciano Mazzoccoli 3,Matias Montes 2ORCID,Giovanni Nigita 4ORCID,Joe T. Sharick 5,Jennifer L. Leight 5ORCID,Federica Calore 1,4,Dawn S. Chandler 2ORCID andRaphael E. Pollock 1,*ORCID

Journal: MDPI (2024)

Abstract

Retroperitoneal liposarcoma (RPLPS) is one of the most common histologic subtypes of soft tissue sarcoma (STS). Complete surgical resection remains the mainstay treatment, while the high rate of locoregional recurrence constitutes the predominant cause of mortality. Well-differentiated (WDLPS) and dedifferentiated (DDLPS) liposarcoma are the most frequent subtypes of RPLPS and present amplified MDM2 gene as a hallmark. However, there are few reports evaluating the role of alternatively spliced MDM2 transcripts in RPLPS. In this study, we assessed MDM2-ALT2 expression levels in a cohort of RPLPS patients and evaluated the biological functions of the MDM2-ALT2 isoform in vitro in DDLPS cell lines. Using BaseScope™ and qPCR, we demonstrated that MDM2-Full Length (MDM2-FL) and MDM2-ALT2 expression levels were upregulated in RPLPS patient-derived tissue samples compared to normal adjacent to tumor tissue (NAT). DDLPS cells overexpressing MDM2-FL or MDM2-ALT2 had higher proliferation rates and increased migration and invasion capacities, as well as increased protein levels of p-AKT, mTOR, p70S6K, MMP2, and cJun. Simultaneous overexpression of MDM2-ALT2 and AKT silencing showed that AKT inhibition impaired p-p70S6K and MMP2 protein increased levels and led to significantly decreased proliferation and migration rates compared to cells overexpressing MDM2-ALT2 only. Taken together, our data suggest that MDM2-ALT2 may promote RPLPS progression.

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Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma

NPJ Precis Oncol (2022)

Authors: Safiya Khurshid 1,2,#, Matias Montes 1,2,#, Daniel F Comiskey Jr 1,2, Brianne Shane 1,2, Eleftheria Matsa 1,2, Francesca Jung 1,2, Chelsea Brown 1,2, Hemant Kumar Bid 3, Ruoning Wang 1,2, Peter J Houghton 4, Ryan Roberts 1,2, Frank Rigo 5, Dawn Chandler 1,2,✉

Journal: NPJ Precis Oncol (2022)

Abstract

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.

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SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway

Mol Cancer Res (2020)

AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer

Oncogene (2018)

E2f3 in tumor macrophages promotes lung metastasis

Oncogene (2017)

Authors: Prashant Trikha 1,2,3, Nidhi Sharma 1,2,3, Clarissa Pena 1,2,3, Andreas Reyes 1,2,3, Thierry Pécot 1,2,3, Safiya Khurshid 1,2,3, Maysoon Rawahneh 1,2,3, Julie Moffitt 1,2,3, Julie A Stephens 4, Soledad A Fernandez 4, Michael Ostrowski 5, Gustavo Leone 1,2,3,*

Journal: Oncogene (2017)

Abstract

The Rb-E2F axis is an important pathway involved in cell cycle control that is deregulated in a number of cancers. E2f transcription factors have distinct roles in the control of cell proliferation, cell survival and differentiation in a variety of tissues. We have previously shown that E2fs are important downstream targets of a CSF-1 signaling cascade involved in myeloid development. In cancer, tumor associated macrophages (TAMs) are recruited to the tumor stroma in response to cytokines secreted by tumor cells and are believed to facilitate tumor cell invasion and metastasis. Using the MMTV-Polyoma Middle T antigen (PyMT) mouse model of human ductal carcinoma, we show that the specific ablation of E2f3 in TAMs, but not in tumor epithelial cells, attenuates lung metastasis without affecting primary tumor growth. Histological analysis and gene expression profiling suggests that E2f3 does not impact the proliferation or survival of TAMs, but rather controls a novel gene expression signature associated with cytoskeleton rearrangements, cell migration and adhesion. This E2f3-TAM gene expression signature was sufficient to predict cancer recurrence and overall survival of ER-positive breast cancer patients. Interestingly, we find that E2f3b but not E2f3a levels are elevated in TAMs from PyMT mammary glands relative to controls, suggesting a differential role for these isoforms in metastasis. In summary, these findings identify E2f3 as a key transcription factor in TAMs that influences the tumor microenvironment and tumor cell metastasis.

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Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation

Dev Cell (2017)

Authors: Huayang Liu1,2,3,12 ∙ James A. Dowdle1,2,3,12 ∙ Safiya Khurshid1,2,3,12 ∙ Nicholas J. Sullivan1,2,3,12 ∙ Nicholas Bertos8,9 ∙ Komal Rambani1,2,3 ∙ Markus Mair1,2,3 ∙ Piotr Daniel1,2,3 ∙ Esther Wheeler2 ∙ Xing Tang1,2,3 ∙ Kyle Toth1,2,3 ∙ Michael Lause1,2,3 ∙ Markus E. Harrigan1,2,3 ∙ Karl Eiring1,2,3 ∙ Connor Sullivan1,2,3 ∙ Matthew J. Sullivan1,2,3 ∙ Serena W. Chang1,2,3 ∙ Siddhant Srivastava1,2,3 ∙ Joseph S. Conway1,2,3 ∙ Raleigh Kladney1,2,3 ∙ Joseph McElroy4,5 ∙ Sooin Bae1,2,3 ∙ Yuanzhi Lu1,2,3 ∙ Ali Tofigh8,10 ∙ Sadiq M.I. Saleh8,10 ∙ Soledad A. Fernandez4,5 ∙ Jeffrey D. Parvin1,5 ∙ Vincenzo Coppola1,3 ∙ Erin R. Macrae6 ∙ Sarmila Majumder1,3 ∙ Charles L. Shapiro6 ∙ Lisa D. Yee7 ∙ Bhuvaneswari Ramaswamy6 ∙ Michael Hallett8,10 ∙ Michael C. Ostrowski1,3 ∙ Morag Park8,9 ∙ Helen M. Chamberlin2 chamberlin.27@osu.edu ∙ Gustavo Leone1,2,3,11,13 leoneg@musc.edu

Journal: Dev Cell (2017)

Abstract

Mesodermal cells signal to neighboring epithelial cells to modulate their proliferation in both normal and disease states. We adapted a Caenorhabditis elegans organogenesis model to enable a genome-wide mesodermal-specific RNAi screen and discovered 39 factors in mesodermal cells that suppress the proliferation of adjacent Ras pathway-sensitized epithelial cells. These candidates encode components of protein complexes and signaling pathways that converge on the control of chromatin dynamics, cytoplasmic polyadenylation, and translation. Stromal fibroblast-specific deletion of mouse orthologs of several candidates resulted in the hyper-proliferation of mammary gland epithelium. Furthermore, a 33-gene signature of human orthologs was selectively enriched in the tumor stroma of breast cancer patients, and depletion of these factors from normal human breast fibroblasts increased proliferation of co-cultured breast cancer cells. This cross-species approach identified unanticipated regulatory networks in mesodermal cells with growth-suppressive function, exposing the conserved and selective nature of mesodermal-epithelial communication in development and cancer.

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AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis

The Embo Journal (2012)

Authors: Katja Höpker 1, Henning Hagmann, Safiya Khurshid, Shuhua Chen, Pia Hasskamp, Tamina Seeger-Nukpezah, Katharina Schilberg, Lukas Heukamp, Tobias Lamkemeyer, Martin L Sos, Roman K Thomas, Drew Lowery, Frederik Roels, Matthias Fischer, Max C Liebau, Ulrike Resch, Tülay Kisner, Fabian Röther, Malte P Bartram, Roman Ulrich Müller, Francesca Fabretti, Peter Kurschat, Björn Schumacher, Matthias Gaestel, René H Medema, Michael B Yaffe, Bernhard Schermer, H Christian Reinhardt, Thomas Benzing

Journal: The Embo Journal (2012)

Abstract

Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens.

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Putting the brakes on p53-driven apoptosis

Cell Cycle (2012)

Authors: Katja Höpker 1, Henning Hagmann, Safiya Khurshid, Shuhua Chen, Bernhard Schermer, Thomas Benzing, Hans Christian Reinhardt

Journal: Cell Cycle (2012)

Abstract

Following genotoxic stress, cells activate a complex, kinase-based signaling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumor suppressor p53 lies at the heart of this DNA damage response. p53 mediates the transactivation of both cell cycle-regulating and pro-apoptotic clusters of target genes. However, it remains incompletely understood which signaling molecules dictate the choice between these two opposing p53-dependent cellular outcomes. Over recent years, numerous regulatory mechanisms impacting on the cellular outcome of p53 signaling have been described. However, no single dominant mechanism has thus far been identified to regulate the cellular choice between p53-driven apoptosis or senescence. The transcriptional regulator AATF has recently emerged as a novel factor impacting on the cellular outcome of the p53 response. Upon genotoxic stress, cytoplasmic pools of MRLC-bound AATF are phosphorylated through the p38MAPK/MK2 checkpoint kinase complex. This AATF phosphorylation results in the disruption of cytoplasmic MRLC3:AATF complexes followed by rapid nuclear localization of AATF. Once in the nucleus, AATF binds to the PUMA, BAX and BAK promoters to repress the DNA damage-induced expression of these pro-apoptotic p53 target genes. Depletion of AATF in tumor cells results in a dramatically enhanced response to DNA-damaging chemotherapeutics, both in vitro and in vivo. Furthermore, focal copy number gains at the AATF locus in neuroblastoma correlate with adverse prognosis and reduced overall survival in this typically p53-proficient malignancy. These data identify the p38/MK2/AATF signaling pathway as a critical repressor of p53-driven apoptosis in tumor cells and implicate this signaling cascade as a novel target for chemotherapy-sensitizing therapeutic efforts.

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Khurshid Lab

About Khurshid Lab

Our Philosophy

Primary Research Focus

Research Areas

Analyze Patient Data to Map the Splicing Landscape in Pediatric Cancer samples

Elucidate alternative splicing deregulation in tumor cells under metabolic starvation and design SSOs to restore them

Investigate the Role of RNA-Binding Proteins (RBPs) in Metabolic Starvation

Publications

Employing splice-switching oligonucleotides and AAVrh74.U7 snRNA to target insulin receptor splicing and cancer hallmarks in osteosarcoma

Blockade of Interleukin-6 (IL-6) Signaling in Dedifferentiated Liposarcoma (DDLPS) Decreases Mouse Double Minute 2 (MDM2) Oncogenicity via Alternative Splicing

Oncogenic Functions of Alternatively Spliced MDM2-ALT2 Isoform in Retroperitoneal Liposarcoma

Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway

AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer

E2f3 in tumor macrophages promotes lung metastasis

Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation

AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis

Putting the brakes on p53-driven apoptosis

Our Team

Dr Safiya Khurshid

Moin Talukder

Naira Rashid

Contact

Khurshid Lab

Khurshid Lab

About Khurshid Lab

Our Philosophy

Primary Research Focus

Research Areas

Analyze Patient Data to Map the Splicing Landscape in Pediatric Cancer samples

Analyze Patient Data to Map the Splicing Landscape in Pediatric Cancer samples

Elucidate alternative splicing deregulation in tumor cells under metabolic starvation and design SSOs to restore them

Elucidate alternative splicing deregulation in tumor cells under metabolic starvation and design SSOs to restore them

Investigate the Role of RNA-Binding Proteins (RBPs) in Metabolic Starvation

Investigate the Role of RNA-Binding Proteins (RBPs) in Metabolic Starvation

Publications

Employing splice-switching oligonucleotides and AAVrh74.U7 snRNA to target insulin receptor splicing and cancer hallmarks in osteosarcoma

Employing splice-switching oligonucleotides and AAVrh74.U7 snRNA to target insulin receptor splicing and cancer hallmarks in osteosarcoma

Abstract

Blockade of Interleukin-6 (IL-6) Signaling in Dedifferentiated Liposarcoma (DDLPS) Decreases Mouse Double Minute 2 (MDM2) Oncogenicity via Alternative Splicing

Blockade of Interleukin-6 (IL-6) Signaling in Dedifferentiated Liposarcoma (DDLPS) Decreases Mouse Double Minute 2 (MDM2) Oncogenicity via Alternative Splicing

Abstract

Oncogenic Functions of Alternatively Spliced MDM2-ALT2 Isoform in Retroperitoneal Liposarcoma

Oncogenic Functions of Alternatively Spliced MDM2-ALT2 Isoform in Retroperitoneal Liposarcoma

Abstract

Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma

Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma

Abstract

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway

SRSF2 Regulation of MDM2 Reveals Splicing as a Therapeutic Vulnerability of the p53 Pathway

Abstract

AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer

AATF suppresses apoptosis, promotes proliferation and is critical for Kras-driven lung cancer

Abstract

E2f3 in tumor macrophages promotes lung metastasis

E2f3 in tumor macrophages promotes lung metastasis

Abstract

Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation

Discovery of Stromal Regulatory Networks that Suppress Ras-Sensitized Epithelial Cell Proliferation

Abstract

AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis

AATF/Che-1 acts as a phosphorylation-dependent molecular modulator to repress p53-driven apoptosis

Abstract

Putting the brakes on p53-driven apoptosis

Putting the brakes on p53-driven apoptosis

Abstract

Welcome

About Khurshid Lab

Our Team

Dr Safiya Khurshid

Dr Safiya Khurshid

Moin Talukder

Moin Talukder

Naira Rashid

Naira Rashid

Contact

Khurshid Lab