modeling-brain-metastases-through-intracranial-injection-magnetic

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Systems biology perspectives are crucial for understanding the pathophysiology of complex diseases, and therefore hold great promise for the discovery of novel treatment strategies. Drug combinations have been shown to improve durability and reduce resistance to available first-line therapies in a variety of cancers; however, traditional drug discovery approaches are prohibitively cost and labor-intensive to evaluate large-scale matrices of potential drug combinations. Computational methods are needed to efficiently model complex interactions of drug target pathways and identify mechanisms underlying drug combination synergy. In this study, we employ a computational approach, SynGeNet (Synergy from Gene expression and Network mining), which integrates transcriptomics-based connectivity mapping and network centrality analysis to analyze disease networks and predict drug combinations. As an exemplar of a disease in which combination therapies demonstrate efficacy in genomic-specific contexts, we investigate malignant melanoma. We employed SynGeNet to generate drug combination predictions for each of the four major genomic subtypes of melanoma (BRAF, NRAS, NF1, and triple wild type) using publicly available gene expression and mutation data. We validated synergistic drug combinations predicted by our method across all genomic subtypes using results from a high-throughput drug screening study across. Finally, we prospectively validated the drug combination for -mutant melanoma that was top ranked by our approach, vemurafenib (BRAF inhibitor) + tretinoin (retinoic acid receptor agonist), using both in vitro and in vivo models of -mutant melanoma and RNA-sequencing analysis of drug-treated melanoma cells to validate the predicted mechanisms. Our approach is applicable to a wide range of disease domains, and, importantly, can model disease-relevant protein subnetworks in precision medicine contexts.

Synergy from gene expression and network mining (SynGeNet) method predicts synergistic drug combinations for diverse melanoma genomic subtypes.

Proneural glioblastoma is defined by an expression pattern resembling that of oligodendrocyte progenitor cells and carries a distinctive set of genetic alterations. Whether there is a functional relationship between the proneural phenotype and the associated genetic alterations is unknown. To evaluate this possible relationship, we performed a longitudinal molecular characterization of tumor progression in a mouse model of proneural glioma. In this setting, the tumors acquired remarkably consistent genetic deletions at late stages of progression, similar to those deleted in human proneural glioblastoma. Further investigations revealed that p53 is a master regulator of the transcriptional network underlying the proneural phenotype. This p53-centric transcriptional network and its associated phenotype were observed at both the early and late stages of progression, and preceded the proneural-specific deletions. Remarkably, deletion of p53 at the time of tumor initiation obviated the acquisition of later deletions, establishing a link between the proneural transcriptional network and the subtype-specific deletions selected during glioma progression.

The transcriptional regulatory network of proneural glioma determines the genetic alterations selected during tumor progression.

The Jak/STAT pathway is activated in human pancreatic ductal adenocarcinoma (PDAC) and cooperates with mutant Kras to drive initiation and progression of PDAC in murine models. We hypothesized that the small-molecule Jak2 inhibitor (BMS-911543) would elicit anti-tumor activity against PDAC and decrease immune suppressive features of the disease. We used an aggressive genetically engineered PDAC model with mutant KrasG12D, tp53R270H, and Brca1 alleles (KPC-Brca1 mice). Mice with confirmed tumor burden were treated orally with vehicle or 30 mg/kg BMS-911543 daily for 14 days. Histologic analysis of pancreata from treated mice revealed fewer foci of adenocarcinoma and significantly decreased Ki67+ cells versus controls. In vivo administration of BMS-911543 significantly reduced pSTAT5 and FoxP3 positive cells within the pancreas, but did not alter STAT3 phosphorylation. Continuous dosing of KPC-Brca1 mice with BMS-911543 resulted in a median survival of 108 days, as compared to a median survival of 87 days in vehicle treated animals, a 23% increase (p = 0.055). In vitro experiments demonstrated that PDAC cell lines were poorly sensitive to BMS-911543, requiring high micromolar concentrations to achieve targeted inhibition of Jak/STAT signaling. Similarly, BMS-911543 had little in vitro effect on the viability of both murine and human PDAC-derived stellate cell lines. However, BMS-911543 potently inhibited phosphorylation of pSTAT3 and pSTAT5 at low micromolar doses in human PBMC and reduced in vitro differentiation of Foxp3+ T regulatory cells. These results indicate that single agent Jak2i deserves further study in preclinical models of PDAC and has distinct inhibitory effects on STAT5 mediated signaling. 

Single agent BMS-911543 Jak2 inhibitor has distinct inhibitory effects on STAT5 signaling in genetically engineered mice with pancreatic cancer.

The contribution of the microenvironment to pancreatic acinar-to-ductal metaplasia (ADM), a preneoplastic transition in oncogenic Kras-driven pancreatic cancer progression, is currently unclear. Here we show that disruption of paracrine Hedgehog signaling via genetic ablation of Smoothened (Smo) in stromal fibroblasts in a Kras(G12D) mouse model increased ADM. Smo-deleted fibroblasts had higher expression of transforming growth factor-α (Tgfa) mRNA and secreted higher levels of TGFα, leading to activation of EGFR signaling in acinar cells and increased ADM. The mechanism involved activation of AKT and noncanonical activation of the GLI family transcription factor GLI2. GLI2 was phosphorylated at Ser230 in an AKT-dependent fashion and directly regulated Tgfa expression in fibroblasts lacking Smo Additionally, Smo-deleted fibroblasts stimulated the growth of Kras(G12D)/Tp53(R172H) pancreatic tumor cells in vivo and in vitro. These results define a non-cell-autonomous mechanism modulating Kras(G12D)-driven ADM that is balanced by cross-talk between Hedgehog/SMO and AKT/GLI2 pathways in stromal fibroblasts.

Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia.

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin-positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a Kras(G12D)-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2-deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras(G12D) signaling during the initial stages of tumor development.

Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia.

Limited efficacy of immune checkpoint inhibitors in pancreatic ductal adenocarcinoma (PDAC) has prompted investigation into combination therapy. We hypothesised that interleukin 6 (IL-6) blockade would modulate immunological features of PDAC and enhance the efficacy of anti-programmed death-1-ligand 1 (PD-L1) checkpoint inhibitor therapy.

IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer.

Senescent cells within the tumor microenvironment (TME) adopt a proinflammatory, senescence-associated secretory phenotype (SASP) that promotes cancer initiation, progression, and therapeutic resistance. Here, exposure to palbociclib (PD-0332991), a CDK4/6 inhibitor, induces senescence and a robust SASP in normal fibroblasts. Senescence caused by prolonged CDK4/6 inhibition is DNA damage-independent and associated with Mdm2 downregulation, whereas the SASP elicited by these cells is largely reliant upon NF-κB activation. Based upon these observations, it was hypothesized that the exposure of nontransformed stromal cells to PD-0332991 would promote tumor growth. Ongoing clinical trials of CDK4/6 inhibitors in melanoma prompted a validation of this hypothesis using a suite of genetically defined melanoma cells (i.e.,  mutant,  mutant, and  wild-type). When cultured in the presence of CDK4/6i-induced senescent fibroblasts, melanoma cell lines exhibited genotype-dependent proliferative responses. However, , PD-0332991-treated fibroblasts enhanced the growth of all melanoma lines tested and promoted the recruitment of Gr-1-positive immune cells. These data indicate that prolonged CDK4/6 inhibitor treatment causes normal fibroblasts to enter senescence and adopt a robust SASP. Such senescent cells suppress the antitumor immune response and promote melanoma growth in immunocompetent,  models. The ability of prolonged CDK4/6 inhibitor treatment to induce cellular senescence and a robust SASP in primary cells may hinder therapeutic efficacy and promote long-term, gerontogenic consequences that should be considered in clinical trials aiming to treat melanoma and other cancer types. .

Stromal Senescence By Prolonged CDK4/6 Inhibition Potentiates Tumor Growth.

Selinexor, a selective inhibitor of nuclear export (SINE) compound targeting exportin-1, has previously been shown to inhibit melanoma cell growth  We hypothesized that combining selinexor with antibodies that block or disrupt T-cell checkpoint molecule signaling would exert superior antimelanoma activity. , selinexor increased  and  gene expression in leukocytes and induced  gene expression in human melanoma cell lines. Mice bearing syngeneic B16F10 melanoma tumors demonstrated a significant reduction in tumor growth rate in response to the combination of selinexor and anti-PD-1 or anti-PD-L1 antibodies ( < 0.05). Similar results were obtained in B16F10-bearing mice treated with selinexor combined with anti-CTLA4 antibody. Immunophenotypic analysis of splenocytes by flow cytometry revealed that selinexor alone or in combination with anti-PD-L1 antibody significantly increased the frequency of both natural killer cells ( ≤ 0.050) and CD4 T cells with a Th1 phenotype ( ≤ 0.050). Further experiments indicated that the antitumor effect of selinexor in combination with anti-PD-1 therapy persisted under an alternative dosing schedule but was lost when selinexor was administered daily. These data indicate that the efficacy of selinexor against melanoma may be enhanced by disrupting immune checkpoint activity. .

The Exportin-1 Inhibitor Selinexor Exerts Superior Antitumor Activity when Combined with T-Cell Checkpoint Inhibitors.

Sex as a biological variable in response to temozolomide.

Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes.

MAX Mutations in Endometrial Cancer: Clinicopathologic Associations and Recurrent MAX p.His28Arg Functional Characterization.

Peritoneal metastasis (PM) is a debilitating consequence of multiple cancers. As cancer cells lose tonic signaling related to attachment dependence, critical morphologic shifts result in alteration of the transcriptome. Identifying key genes associated with this transformation may lead to targeted therapies for this devastating complication. TC71, CHLA9, PANC1, YOU and HEYA8 cell lines were grown as tumor spheroids in polyHEMA coated plates. Temporal profiling of transcriptomic alterations over 72 hrs was used to develop a comprehensive PM model. We identified transcriptomic outliers using Gaussian mixtures model clustering to identify drivers of spheroid formation. Outliers were validated in The Cancer Genome Atlas (TCGA) and an ovarian tissue microarray (TMA) and by modulation in ovarian cancer models  and in peritoneal xenograft models. Outlier analysis of PM genes identified the gene TXNIP and the TORC signaling as central to PM. Ovarian cancer spheroids isolated from patient ascites had significantly higher TXNIP than their attached counterparts ( = 0.047). TXNIP levels predicted progression-free (log-rank  = 0.026) survival in stage 1/2 ovarian cancer and overall survival (log rank  = 0.047) in stage 3/4 ovarian cancer. , TXNIP silencing was associated with increased mTOR signaling and enhanced spheroid development which could be overcome by TAK228, a TORC1/2 inhibitor. Similarly,  peritoneal xenograft models of carcinomatosis were prevented by TAK228. PM is driven by TXNIP-associated TORC1/2 signaling. This work provides the first evidence that TORC1/2 inhibition may prevent PM.

Loss of TXNIP enhances peritoneal metastasis and can be abrogated by dual TORC1/2 inhibition.

Dedifferentiated liposarcomas (DDLPS) are mesenchymal tumors associated with universally poor response to treatment. Genomic amplification of murine double minute 2 () is used as a diagnostic biomarker; however, no established biomarkers exist to guide DDLPS treatment. In the largest study of its kind, we report that the extent of  amplification, not simply the presence of  amplification, may be biologically important to the actions of DDLPS.

Degree of  Amplification Affects Clinical Outcomes in Dedifferentiated Liposarcoma.

Cachexia is a wasting syndrome characterized by pronounced skeletal muscle loss. In cancer, cachexia is associated with increased morbidity and mortality and decreased treatment tolerance. Although advances have been made in understanding the mechanisms of cachexia, translating these advances to the clinic has been challenging. One reason for this shortcoming may be the current animal models, which fail to fully recapitulate the etiology of human cancer-induced tissue wasting. Because pancreatic ductal adenocarcinoma (PDA) presents with a high incidence of cachexia, we engineered a mouse model of PDA that we named KPP. KPP mice, similar to PDA patients, progressively lose skeletal and adipose mass as a consequence of their tumors. In addition, KPP muscles exhibit a similar gene ontology as cachectic patients. We envision that the KPP model will be a useful resource for advancing our mechanistic understanding and ability to treat cancer cachexia.

Modeling Human Cancer-induced Cachexia.

Dedifferentiated liposarcoma (DDLPS) is a highly morbid mesenchymal tumor characterized and driven by genomic amplification of the  gene. Direct inhibition of MDM2 has shown promise pre-clinically, but has yet to be validated in clinical trials. Early  studies have demonstrated that pan-histone deacetylase (HDAC) inhibition may have anti-MDM2 effects. Here we present , , and mouse xenograft studies that suggest that specifically targeting HDAC2 reduces MDM2 expression and has anti-tumor affects in DDLPS. Two independent datasets, The Cancer Genome Atlas (TCGA;  = 58) and the Memorial Sloan-Kettering Cancer Center Dataset (MSKCC;  = 63), were used to identify the co-expression between class I  and , and their clinical impact. 2 was highly co-expressed with  (TCGA: Spearman's coefficient = 0.29,  = 0.03; MSKCC: Spearman's coefficient = 0.57,  < 0.001). As both a continuous and dichotomous predictor, elevated  expression was associated with worsened disease-free survival in the TCGA (Continuous: Hazard-ratio (HR) 1.7; 95% Confidence Interval (95%CI) 0.97-2.9;  = 0.06; Dichotomous: HR 7.1, 95%CI 2.5-19.8,  < 0.001) and distant recurrence-free survival in the MSKCC (Continuous: HR 2.2; 95%CI 1.1-4.8;  = 0.04; Dichotomous: HR 2.8, 95%CI 1.2-6.4,  = 0.02). , treatment of DDLPS cell lines with the HDAC inhibitors MI-192 (HDAC2/3 inhibitor) or romidepsin (HDAC1/2 inhibitor) reduced  expression and induced apoptosis. In a murine DDLPS xenograft model, romidepsin reduced tumor growth and lowered tumor MDM2 expression. RNA-sequencing of romidepsin treated mouse tumors demonstrated markers of TP53 reactivation. Taken together, our data supports the hypothesis that targeting HDAC2 may represent a potential strategy to modulate MDM2 expression in DDLPS.

Inhibition of histone deacetylase 2 reduces MDM2 expression and reduces tumor growth in dedifferentiated liposarcoma.

Alterations of the PALB2 tumor suppressor gene have been identified in familial breast, ovarian and pancreatic cancer cases. PALB2 cooperates with BRCA1/2 proteins through physical interaction in initiation of homologous recombination, in maintenance of genome integrity following DNA double-strand breaks. To determine if the role of PALB2 as a linker between BRCA1 and BRCA2 is critical for BRCA1/2-mediated tumor suppression, we generated Palb2 mouse pancreatic cancer models and compared tumor latencies, phenotypes and drug responses with previously generated Brca1/2 pancreatic cancer models. For development of Palb2 pancreatic cancer, we crossed conditional Palb2 null mouse with mice carrying the KrasG12D; p53R270H; Pdx1-Cre (KPC) constructs, and these animals were observed for pancreatic tumor development. Individual deletion of Palb2, Brca1 or Brca2 genes in pancreas per se using Pdx1-Cre was insufficient to cause tumors, but it reduced pancreata size. Concurrent expression of mutant KrasG12D and p53R270H, with tumor suppressor inactivated strains in Palb2-KPC, Brca1-KPC or Brca2-KPC, accelerated pancreatic ductal adenocarcinoma (PDAC) development. Moreover, most Brca1-KPC and some Palb2-KPC animals developed mucinous cystic neoplasms with PDAC, while Brca2-KPC and KPC animals did not. 26% of Palb2-KPC mice developed MCNs in pancreata, which resemble closely the Brca1 deficient tumors. However, the remaining 74% of Palb2-KPC animals developed PDACs without any cysts like Brca2 deficient tumors. In addition, the number of ADM lesions and immune cells infiltrations (CD3+ and F/480+) were significantly increased in Brca1-KPC tumors, but not in Brca2-KPC tumors. Interestingly, the level of ADM lesions and infiltration of CD3+ or F/480+ cells in Palb2-KPC tumors were intermediate between Brca1-KPC and Brca2-KPC tumors. As expected, disruption of Palb2 and Brca1/2 sensitized tumor cells to DNA damaging agents in vitro and in vivo. Altogether, Palb2-KPC PDAC exhibited features observed in both Brca1-KPC and Brca2-KPC tumors, which could be due to its role, as a linker between Brca1 and Brca2.

Murine models for familial pancreatic cancer: Histopathology, latency and drug sensitivity among cancers of Palb2, Brca1 and Brca2 mutant mouse strains.

Article	Year
Synergy from gene expression and network mining (SynGeNet) method predicts synergistic drug combinations for diverse melanoma genomic subtypes. NPJ systems biology and applications\| PubMed ID: 30820351	2019
The transcriptional regulatory network of proneural glioma determines the genetic alterations selected during tumor progression. Cancer research\| PubMed ID: 24390738	2014
Single agent BMS-911543 Jak2 inhibitor has distinct inhibitory effects on STAT5 signaling in genetically engineered mice with pancreatic cancer. Oncotarget\| PubMed ID: 26575024	2015
Genetic ablation of Smoothened in pancreatic fibroblasts increases acinar-ductal metaplasia. Genes & development\| PubMed ID: 27633013	2016
Stromal ETS2 Regulates Chemokine Production and Immune Cell Recruitment during Acinar-to-Ductal Metaplasia. Neoplasia (New York, N.Y.)\| PubMed ID: 27659014	2016
IL-6 and PD-L1 antibody blockade combination therapy reduces tumour progression in murine models of pancreatic cancer. Gut\| PubMed ID: 27797936	2016
Stromal Senescence By Prolonged CDK4/6 Inhibition Potentiates Tumor Growth. Molecular cancer research : MCR\| PubMed ID: 28039358	2017
The Exportin-1 Inhibitor Selinexor Exerts Superior Antitumor Activity when Combined with T-Cell Checkpoint Inhibitors. Molecular cancer therapeutics\| PubMed ID: 28148715	2017
Sex as a biological variable in response to temozolomide. Neuro-oncology\| PubMed ID: 28379437	2017
MAX Mutations in Endometrial Cancer: Clinicopathologic Associations and Recurrent MAX p.His28Arg Functional Characterization. Journal of the National Cancer Institute\| PubMed ID: 29155953	2018
Loss of TXNIP enhances peritoneal metastasis and can be abrogated by dual TORC1/2 inhibition. Oncotarget\| PubMed ID: 30479697	2018
Degree of Amplification Affects Clinical Outcomes in Dedifferentiated Liposarcoma. The oncologist\| PubMed ID: 31019022	2019
Modeling Human Cancer-induced Cachexia. Cell reports\| PubMed ID: 31390573	2019
Inhibition of histone deacetylase 2 reduces MDM2 expression and reduces tumor growth in dedifferentiated liposarcoma. Oncotarget\| PubMed ID: 31620242	2019
Murine models for familial pancreatic cancer: Histopathology, latency and drug sensitivity among cancers of Palb2, Brca1 and Brca2 mutant mouse strains. PloS one\| PubMed ID: 31877165	2019

Reena Shakya

Biography

JoVE Journal Publications

Other Publications