Biology subjects

Pratilas, C. A.

Publications and source records attributed to Pratilas, C. A..

11 recordsLinked to original sources

bioRxiv2026

Integrated ex vivo screening and transcriptomic profiling to prioritize drug combinations for rare cancers

Discovering effective drug combinations requires testing many dose combinations across a diverse panel of tumor models. This approach is limited in rare cancers by a scarcity of cell lines and representative high-throughput models that would make exhaustive screening feasible and predictive. Patient-derived xenograft (PDX) models are genomically representative but too low-throughput for this purpose. Culturing PDX cells ex vivo in three-dimensional (3D) matrices offers a genomically representative and clinically relevant platform for preclinical drug testing, capturing the microenvironmental cues that shape in vivo drug response while requiring only limited tissue per assay. Toward this end, we designed and validated an experimental-computational framework, "ex vivo assessment of combination therapies" (EXACT), to enable drug combination discovery in rare tumors. Using PDX models of malignant peripheral nerve sheath tumors (MPNST), we built a platform to culture PDX cells ex vivo over multiple days, monitoring drug sensitivity and measuring transcriptomic responses to treatment. Computational analysis of these transcriptomic responses then identifies which compensatory pathway creates a unique vulnerability to a second drug. EXACT thus offers a biologically informed, scalable approach for prioritizing drug combinations in rare tumors, nominating drugs alongside biological rationale. Using this methodology, we identified a MEK inhibitor plus HDAC inhibitor combination with enhanced activity in vitro and in vivo, forming the basis of an active clinical trial. This platform could be adapted for real-time use with primary patient specimens, enabling personalized therapeutic discovery. SignificanceEXACT integrates PDX-derived 3D drug screening with biologically informed computational analysis to identify and explain effective combinations, providing a scalable strategy for therapeutic discovery in rare cancers such as MPNST.

cancer biology↗

bioRxiv2026

ZNF423 depletion induces the integrated stress response and represents a potential vulnerability in NF1-associated MPNST

Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas with limited systemic therapies and represent the leading cause of mortality for individuals with neurofibromatosis type 1 (NF1). Malignant progression can reactivate developmental precursor programs that are largely absent from normal nerve and benign tumors, creating tumor-selective vulnerabilities. Zinc finger protein 423 (ZNF423; also known as OAZ/ROAZ) is a developmentally regulated transcription factor that delays olfactory precursor differentiation and has been implicated in B-cell malignancy. Here, we asked whether ZNF423 is reactivated and functionally required in NF1-associated MPNST. In genetically defined models, Nf1 loss reduced Zfp423 in a benign tumor cell-of-origin context, whereas combined Nf1 and Cdkn2a loss induced marked Zfp423 upregulation during transformation. ZNF423 depletion impaired DNA synthesis and proliferation, induced DNA damage signaling, and activated the integrated stress response (ISR), increasing sensitivity to cytotoxic agents. In an orthotopic MPNST model, shRNA-mediated suppression of ZNF423 reduced tumor initiation in vivo; however, tumors that eventually emerged showed restoration of ZNF423 expression. ZNF423 is developmentally restricted in the peripheral nerve lineage yet elevated in MPNST, with single-cell analyses of patient nerve sheath tumors revealing localized expression restricted to malignant cells rather than SOX10-positive benign tumor cells. These data identify ZNF423 as a putative malignant biomarker, a potential dependency in NF1-MPNST, and nominate downstream stress and genome maintenance pathways as cooperative therapeutic vulnerabilities. STATEMENT OF SIGNIFICANCEZNF423 is a developmentally restricted transcription factor selectively reactivated in NF1-associated malignant peripheral nerve sheath tumors. Targeted ablation triggers the integrated stress response, impairs DNA synthesis, sensitizes cells to chemotherapy and PARP inhibition, and restricts in vivo growth. ZNF423 represents a candidate biomarker and therapeutic vulnerability in this aggressive sarcoma.

cancer biology↗

bioRxiv2026

Multi-omics integration of malignant peripheral nerve sheath tumors identifies potential targets based on chromosome 8q status

BackgroundChromosome 8q (chr8q) copy number gain is associated with high-grade transformation in malignant peripheral nerve sheath tumors (MPNST), an aggressive soft tissue tumor with poor outcomes in the high-risk and metastatic settings. Although chr8q gain is associated with inferior overall survival in patients with MPNST, standard of care therapies do not currently consider stratification by genomic features, including chr8q status. MethodsWe employed a proteogenomic approach to characterize proteomic and transcriptional programs associated with chr8q and nominate drug targets for potential treatment stratification based on chr8q status. We leveraged our growing library of fully characterized MPNST patient-derived xenografts (PDX) and collected LC-MS/MS global and phospho-proteomics measurements for six of these samples. We then integrated these data with transcriptomics and copy number data to identify molecular changes that are correlated with chr8q copy number. We nominated pathways, transcription factors, and kinases that were differentially active in chr8q gain samples and posited that these samples would respond differently to drugs compared to chr8q wildtype samples. We then tested this hypothesis in vitro. ResultsOur results suggest that the chr8q gene MYC may be a key driver of downstream effects that can be targetable with inhibitors of PLK1. Conversely, EGFR inhibition may be more effective in MYC-diploid MPNSTs than those with MYC gain. These results nominate candidate pathways and drug classes to target tumor heterogeneity in MPNST through the proteogenomic integration and drug sensitivity prediction in distinct tumor subpopulations. ConclusionsWe show that integration of multiomics data can identify specific drug therapies to selectively target tumor cells based on chr8q copy number. This not only provides novel avenues for drug nomination going forward but also may be important for stratifying treatment and mitigating resistance in heterogeneous tumors.

cancer biology↗

bioRxiv2024

Combined inhibition of SHP2 overcomes adaptive resistance to type 1 BRAF inhibitors in BRAF V600E-driven high-grade glioma

BRAF-mutant gliomas can be therapeutically targeted with BRAF mutant-selective inhibitors, yet responses are often transient due to short-term adaptive or long-term treatment-emergent resistance. We hypothesized that vertical inhibition of multiple signaling nodes could improve the durability of BRAF inhibition and prevent or overcome adaptive resistance. Using human tissue samples, we identified frequent RAS pathway reactivation in gliomas resistant to BRAF inhibitors, suggesting a common escape mechanism. Using patient-derived cell lines, we observed that upregulation of RAS activity was an adaptive response to BRAFi and that knockdown of SHP2, a central regulator of RAS activity, resulted in enhanced sensitivity to BRAF or MEK inhibition. Moreover, combined small molecule inhibition with SHP2 and BRAF or MEK inhibitors increased the depth and durability of ERK pathway inhibition, as well as prevented paradoxical upregulation of RAS activity. RNA sequencing analysis revealed deeper suppression of ERK transcriptional output with combined therapy, along with decreased reactivation of EGFR. Combined SHP2/BRAF small molecule inhibitors prevented growth and induced cell death in some cell line models. In cell lines with treatment-emergent resistance, moreover, combined SHP2 and BRAF inhibition overcame resistance to BRAF inhibitor monotherapy. In vivo orthotopic and patient-derived xenograft models confirmed enhanced tumor growth inhibition with combined therapy. Together, our findings demonstrate the critical role of RAS/ERK signaling reactivation in driving resistance to BRAF inhibition in glioma, and demonstrate the potential utility for adding SHP2 inhibitors to overcome resistance in BRAF V600E mutant glioma. SignificanceThe addition of a SHP2i to BRAFi in BRAF-V600E glioma cells prevents tumor growth and can overcome resistance to BRAFi in preclinical models in vitro and in vivo.

cancer biology↗

bioRxiv2024

Genomic features of NF1-associated peripheral nerve sheath tumors: a cohort analysis from the Johns Hopkins NF1 biospecimen repository

Neurofibromatosis type 1 (NF1) is an inherited neurocutaneous condition that predisposes to the development of peripheral nerve sheath tumors (PNST) including cutaneous neurofibromas (CNF), plexiform neurofibromas (PNF), atypical neurofibromatous neoplasms with unknown biological potential (ANNUBP), and malignant peripheral nerve sheath tumors (MPNST). The successful advancement of therapeutic development for NF1-associated PNST necessitates the systematic acquisition and analysis of human tumor specimens and their corresponding model systems. RNA sequencing (RNAseq) and whole exome sequencing (WES) data were generated from 73 and 114 primary human tumor samples, respectively. These pre-processed data, standardized for immediate computational analysis, are accessible through the NF Data Portal, allowing immediate interrogation. This analysis combines new and previously released samples, offering a comprehensive view of the entire cohort sequenced. As a dedicated effort to systematically bank tumor samples from people with NF1, in collaboration with molecular geneticists and computational biologists to advance understanding of NF1 biology, the Johns Hopkins NF1 biospecimen repository offers access to samples and genomic data to promote advancement of NF1-related therapies.

bioinformatics↗

bioRxiv2023

MEK inhibition enhances the antitumor effect of radiation therapy in NF1-deficient glioblastoma

Individuals with neurofibromatosis type 1 (NF-1), an autosomal dominant neurogenetic and tumor predisposition syndrome, are susceptible to developing low-grade glioma (LGG) and, less commonly, high-grade glioma (HGG). These gliomas exhibit loss of the neurofibromin gene (NF1), and 10-15% of sporadic HGG have somatic NF1 alterations. Loss of NF1 leads to hyperactive RAS signaling, creating opportunity given the established efficacy of MEK inhibitors (MEKi) in plexiform neurofibromas and some individuals with LGG. We observed that NF1-deficient glioblastoma neurospheres were sensitive to the combination of a MEKi (mirdametinib) with irradiation, as evidenced by synergistic inhibition of cell growth, colony formation, and increased cell death. In contrast, NF1-intact neurospheres were not sensitive to the combination, despite complete ERK pathway inhibition. No neurosphere lines exhibited enhanced sensitivity to temozolomide combined with mirdametinib. Mirdametinib decreased transcription of homologous recombination genes and RAD51 foci, associated with DNA damage repair, in sensitive models. Heterotopic xenograft models displayed synergistic growth inhibition to mirdametinib combined with irradiation in NF1-deficient glioma xenografts, but not those with intact NF1. In sensitive models, benefits were observed at least three weeks beyond the completion of treatment, including sustained phospho-ERK inhibition on immunoblot and decreased Ki-67 expression. These observations demonstrate synergistic activity between mirdametinib and irradiation in NF1-deficient glioma models and may have clinical implications for patients with gliomas that harbor germline or somatic NF1 alterations.

cancer biology↗

bioRxiv2023

CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon restoration of RB function in malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumors (MPNST) are highly aggressive soft tissue sarcomas with limited treatment options, and novel effective therapeutic strategies are desperately needed. We observe anti-proliferative efficacy of genetic depletion or pharmacological inhibition using the clinically available SHP2 inhibitor (SHP2i) TNO155. Our studies into the signaling response to SHP2i reveal that resistance to TNO155 is partially mediated by reduced RB function, and we therefore test the addition of a CDK4/6 inhibitor (CDK4/6i) to enhance RB activity and improve TNO155 efficacy. In combination, TNO155 attenuates the adaptive response to CDK4/6i, potentiates its anti-proliferative effects, and converges on enhancement of RB activity, with greater suppression of cell cycle and inhibitor-of-apoptosis proteins, leading to deeper and more durable anti-tumor activity in in vitro and in vivo patient-derived models of MPNST, relative to either single agent. Overall, our study provides timely evidence to support the clinical advancement of this combination strategy in patients with MPNST and other tumors driven by loss of NF1.

cancer biology↗

bioRxiv2022

Ex vivo to in vivo model of malignant peripheral nerve sheath tumors for precision oncology

Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas that often develop in patients with neurofibromatosis type 1 (NF1-MPNST), but can occur sporadically. Through a multi-institution collaboration, we have developed 13 NF1-associated MPNST patient-derived xenografts (PDX). Genomic analysis of the PDX-tumor pairs identified somatic mutations in NF1 (61%), SUZ12 (61%), EED (15%), and TP53 (15%), and chromosome 8 (Chr8) gain (77%), consistent with published data. Pre-clinical models that capture this molecular heterogeneity are needed to identify and prioritize effective drug candidates for clinical translation. Here, we describe the successful development of a medium-throughput ex vivo 3D microtissue model with several advantages over 2D cell line growth, which can be utilized to predict drug response in vivo. Herein, we present proof-of-principle of this PDX-to-microtissue system, using four genomically representative MPNST and three drugs. This work highlights the development of a novel ex vivo to in vivo preclinical platform in MPNST that successfully captures the genomic diversity observed in patients and represents a resource to identify future therapeutic strategies.

cancer biology↗

bioRxiv2022

Therapeutic vulnerability to ATR inhibition in concurrent NF1 and ATRX-deficient high-grade solid tumors

Subsets of Neurofibromatosis Type 1 (NF1)-associated solid tumors have been shown to display high frequencies of ATRX mutations and presence of alternative lengthening of telomeres (ALT). In addition, a potential biologic vulnerability of ALT-positive cancer cells is ATR inhibition, a regulator of homologous recombination. We studied the phenotype of combined NF1 and ATRX deficiency in malignant solid tumors. For these experiments, cell lines derived from NF1-deficient sporadic glioblastomas (U251, SF188), a NF1-associated ATRX mutant glioblastoma cell line (JHH-NF1-GBM1), a NF1-derived sarcoma cell line (JHH-CRC65), and two NF1-deficient MPNST cell lines (ST88-14, NF90.8) were utilized. Cancer cells were treated with ATR inhibitors (AZD6738 and VE-822), in vitro, with or without a MEK inhibitor (AZD6244, selumetinib) or temozolomide. In contrast to the glioma cell line SF188, combined ATRX knockout (KO) and TERC KO led to ALT-like properties and sensitized U251 glioma cells to ATR inhibition (AZD6738 and VE-822) in vitro and in vivo. In addition, ATR inhibitors sensitized U251 cells to temozolomide, but not MEK inhibition (AZD6244), irrespective of ATRX level manipulation; whereas, the JHH-NF1-GBM1 cell line (ATRX loss/ALT-positive) demonstrated sensitivity to ATR inhibition (AZD6738), but not temozolomide. Similar effects were noted using the MPNST cell line NF90.8 after combined ATRX knockdown and TERC KO; however, not in the MPNST cell line ST88-14. Taken together, our study supports the feasibility of targeting the ATR pathway in subsets of NF1-deficient and associated tumors. Tumors with pre-existing ALT, or that subsequently develop ALT after ATRX downregulation, are particularly vulnerable to this therapeutic approach.

cancer biology↗

bioRxiv2020

Establishment of multiple novel patient-derived models of desmoplastic small round cell tumor enabling functional characterization of ERBB pathway signaling and pre-clinical evaluation of a novel targeted therapy approach

Desmoplastic small round cell tumor (DSRCT) is characterized by the t(11;22)(p13;q12) chromosomal translocation, which fuses the transcriptional regulatory domain of EWSR1 with the zinc finger DNA-binding domain of WT1, resulting in the oncogenic transcription factor EWS-WT1. DSRCT primarily affects young males and has a 5-year overall survival of about 15%. Typical treatment approaches for patients with DSRCT involve a multi-modal combination of surgery, chemotherapy and radiation. The paucity of DSRCT disease models has hampered functional and pre-clinical therapeutic studies in this aggressive cancer. Here, we developed robust preclinical disease models and mined DSRCT expression profiling data to identify genetic vulnerabilities that could be leveraged for the identification of rational therapies. Specifically, we developed four new DSRCT cell lines and one patient-derived xenograft (PDX) model. Transcriptomic and proteomic profiling showed evidence of activation of the ERBB pathway. Ectopic expression of EWSR1-WT1 resulted in upregulation of ERRB family ligands and downstream signaling. Treatment of DSRCT cell lines with ERBB ligands resulted in activation of EGFR, ERBB2, ERK1/2 and AKT, and stimulation of cell growth. Conversely, targeting of EGFR using shRNA, small molecule inhibitors (afatinib, neratinib) or an anti-EGFR antibody (cetuximab) inhibited growth and induced apoptosis in DSRCT cells. Finally, treatment of mice bearing DSRCT xenografts with a combination of cetuximab and afatinib significantly reduced tumor growth. These data provide a rationale for the clinical evaluation of EGFR antagonists in patients with DSRCT.

cancer biology↗

bioRxiv2019

A clinically and genomically annotated nerve sheath tumor biospecimen repository.

Nerve sheath tumors occur as a heterogeneous group of neoplasms in patients with neurofibromatosis type 1 (NF1). The malignant form represents the most common cause of death in people with NF1, and even when benign, these tumors can result in significant disfigurement, neurologic dysfunction, and a range of profound symptoms. Lack of human tissue across the peripheral nerve tumors common in NF1 has been a major limitation in the development of new therapies. To address this unmet need, we have created an annotated collection of patient tumor samples, patient-derived cell lines, and patient-derived xenografts, and carried out high-throughput genomic and transcriptomic characterization to serve as a resource for further biologic and preclinical therapeutic studies. In this work, we release genomic and transcriptomic datasets comprised of 55 tumor samples derived from 23 individuals, complete with clinical annotation. All data are publicly available through the NF Data Portal and at http://synapse.org/jhubiobank.

cancer biology↗