The SRG Rat, a Sprague-Dawley Rag2/Il2rg Double-Knockout Validated for Human Tumor Oncology Studies

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks

The Role of KLF6 Gene in Lung Cancer

Lung cancer is the leading cause of cancer related death in the United States and the high mortality rate associated with lung cancer has prompted efforts in understanding the molecular origins of the disease. Epidermal growth factor receptor (EGFR) activation is both a key molecular driver of disease progression and the target of a broad class of molecular agents designed to treat advanced cancer. Nevertheless, resistance develops through several mechanisms including constitutive activation of AKT signaling. Additional molecular characterization of the downstream mediators of EGFR signaling may lead to the development of new classes of targeted molecular therapies to treat resistant disease. Here I identify a transcriptional network involving the KLF6 and FOXO1 tumor suppressor genes that negatively regulate activated EGFR signaling and that can be reactivated using the combination of two FDA approved agents in both cell culture and in vivo models of the disease. In both murine models and patient derived lung adenocarcinoma samples, EGFR activation is associated with FOXO1 mislocalization and decreased KLF6 expression. Furthermore, in a Kras driven mouse model, KLF6 expression is not significantly changed whereas AKT activation seen in the Pten/Mmac1+/- heterozygous mouse model results in FOXO1 mislocalization and decreased KLF6 expression. Consistent with these findings, inhibition of AKT signaling promotes increase in nuclear FOXO1 resulting in transactivation of the KLF6 tumor suppressor gene in lung adenocarcinoma cell lines. Correspondingly, the EGFRL858R mouse model demonstrates spontaneous tumor regression when treated with the anti-EGFR based therapy, erlotinib, an FDA-approved small-molecule inhibitor of EGFR signaling. I analyzed L858R mouse tumors samples treated with erlotinib and found increased KLF6 expression following EGFR inhibition. Conversely, targeted reduction of KLF6 resulted in decreased erlotinib response in both cell culture and in vivo models of disease suggesting a direct link between KLF6 upregulation and the induction of apoptosis by anti-EGFR based therapy. Therefore, I hypothesized that acquired resistance to anti-EGFR based therapies could be overcome by restoring downstream function of the FOXO1/KLF6 transcriptional network. Here I demonstrate that an FDA-approved drug, trifluoperazine hydrochloride (TFP), which has been shown to inhibit FOXO1 nuclear export, restores sensitivity to AKT-driven erlotinib-resistance through modulation of the KLF6/FOXO1 signaling cascade in both cell culture and xenograft models. Furthermore, silencing of FOXO1 blunts apoptosis mediated through combination erlotinib and TFP treatment suggesting that this transcriptional network is important for negatively regulating AKT signaling. Combined, these studies define a novel transcriptional network regulating oncogenic EGFR signaling and identify a class of FDA-approved drugs with the potential for rapid clinical translation to restore chemosensitivity to anti-EGFR-based therapy for the treatment of metastatic lung adenocarcinoma. Furthermore the alternative splice variant of the KLF6 tumor suppressor gene, KLF6-SV1, has been shown to be associated with decreased survival in patients with lung adenocarcinoma. Here I demonstrate that lung cancer cells that are chemoresistant express increased levels of KLF6-SV1 and targeted reduction of KLF6-SV1 using RNA interference restores chemosensitivity both in culture and in vivo. These findings highlight a role for KLF6-SV1 in regulation of chemotherapy response

PP2A inhibition is a druggable MEK inhibitor resistance mechanism in KRAS-mutant lung cancer cells

Kinase inhibitor resistance constitutes a major unresolved clinical challenge in cancer. Furthermore, the role of serine/threonine phosphatase deregulation as a potential cause for resistance to kinase inhibitors has not been thoroughly addressed. We characterize protein phosphatase 2A (PP2A) activity as a global determinant of KRAS-mutant lung cancer cell resistance across a library of >200 kinase inhibitors. The results show that PP2A activity modulation alters cancer cell sensitivities to a large number of kinase inhibitors. Specifically, PP2A inhibition ablated mitogen-activated protein kinase kinase (MEK) inhibitor response through the collateral activation of AKT/mammalian target of rapamycin (mTOR) signaling. Combination of mTOR and MEK inhibitors induced cytotoxicity in PP2A-inhibited cells, but even this drug combination could not abrogate MYC up-regulation in PP2A-inhibited cells. Treatment with an orally bioavailable small-molecule activator of PP2A DT-061, in combination with the MEK inhibitor AZD6244, resulted in suppression of both p-AKT and MYC, as well as tumor regression in two KRAS-driven lung cancer mouse models. DT-061 therapy also abrogated MYC-driven tumorigenesis. These data demonstrate that PP2A deregulation drives MEK inhibitor resistance in KRAS-mutant cells. These results emphasize the need for better understanding of phosphatases as key modulators of cancer therapy responses

KLF6-SV1 overexpression accelerates human and mouse prostate cancer progression and metastasis

Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1–overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target

The SRG rat, a Sprague-Dawley Rag2/Il2rg double-knockout validated for human tumor oncology studies.

We have created the immunodeficient SRG rat, a Sprague-Dawley Rag2/Il2rg double knockout that lacks mature B cells, T cells, and circulating NK cells. This model has been tested and validated for use in oncology (SRG OncoRat®). The SRG rat demonstrates efficient tumor take rates and growth kinetics with different human cancer cell lines and PDXs. Although multiple immunodeficient rodent strains are available, some important human cancer cell lines exhibit poor tumor growth and high variability in those models. The VCaP prostate cancer model is one such cell line that engrafts unreliably and grows irregularly in existing models but displays over 90% engraftment rate in the SRG rat with uniform growth kinetics. Since rats can support much larger tumors than mice, the SRG rat is an attractive host for PDX establishment. Surgically resected NSCLC tissue from nine patients were implanted in SRG rats, seven of which engrafted and grew for an overall success rate of 78%. These developed into a large tumor volume, over 20,000 mm3 in the first passage, which would provide an ample source of tissue for characterization and/or subsequent passage into NSG mice for drug efficacy studies. Molecular characterization and histological analyses were performed for three PDX lines and showed high concordance between passages 1, 2 and 3 (P1, P2, P3), and the original patient sample. Our data suggest the SRG OncoRat is a valuable tool for establishing PDX banks and thus serves as an alternative to current PDX mouse models hindered by low engraftment rates, slow tumor growth kinetics, and multiple passages to develop adequate tissue banks