Madhu S. Kumar

The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer deaths worldwide; nearly half contain mutations in the receptor tyrosine kinase/RAS pathway. Here we show that RAS-pathway mutant NSCLC cells depend on the transcription factor GATA2. Loss of GATA2 reduced the viability of NSCLC cells with RAS-pathway mutations, whereas wild-type cells were unaffected. Integrated gene expression and genome occupancy analyses revealed GATA2 regulation of the proteasome, and IL-1-signaling, and Rho-signaling pathways. These pathways were functionally significant, as reactivation rescued viability after GATA2 depletion. In a Kras-driven NSCLC mouse model, Gata2 loss dramatically reduced tumor development. Furthermore, Gata2 deletion in established Kras mutant tumors induced striking regression. Although GATA2 itself is likely undruggable, combined suppression of GATA2-regulated pathways with clinically approved inhibitors caused marked tumor clearance. Discovery of the nononcogene addiction of KRAS mutant lung cancers to GATA2 presents a network of druggable pathways for therapeutic exploitation.

HMGA2 functions as a competing endogenous RNA to promote lung cancer progression

Non-small-cell lung cancer (NSCLC) is the most prevalent histological cancer subtype worldwide. As the majority of patients present with invasive, metastatic disease, it is vital to understand the basis for lung cancer progression. Hmga2 is highly expressed in metastatic lung adenocarcinoma, in which it contributes to cancer progression and metastasis. Here we show that Hmga2 promotes lung cancer progression in mouse and human cells by operating as a competing endogenous RNA (ceRNA) for the let-7 microRNA (miRNA) family. Hmga2 can promote the transformation of lung cancer cells independent of protein-coding function but dependent upon the presence of let-7 sites; this occurs without changes in the levels of let-7 isoforms, suggesting that Hmga2 affects let-7 activity by altering miRNA targeting. These effects are also observed in vivo, where Hmga2 ceRNA activity drives lung cancer growth, invasion and dissemination. Integrated analysis of miRNA target prediction algorithms and metastatic lung cancer gene expression data reveals the TGF-β co-receptor Tgfbr3 (ref. 12) as a putative target of Hmga2 ceRNA function. Tgfbr3 expression is regulated by the Hmga2 ceRNA through differential recruitment to Argonaute 2 (Ago2), and TGF-β signalling driven by Tgfbr3 is important for Hmga2 to promote lung cancer progression. Finally, analysis of NSCLC-patient gene-expression data reveals that HMGA2 and TGFBR3 are coordinately regulated in NSCLC-patient material, a vital corollary to ceRNA function. Taken together, these results suggest that Hmga2 promotes lung carcinogenesis both as a protein-coding gene and as a non-coding RNA; such dual-function regulation of gene-expression networks reflects a novel means by which oncogenes promote disease progression.

Requirement for Interaction of PI3-Kinase p110α with RAS in Lung Tumor Maintenance

Summary RAS proteins directly activate PI3-kinases. Mice bearing a germline mutation in the RAS binding domain of the p110α subunit of PI3-kinse are resistant to the development of RAS-driven tumors. However, it is unknown whether interaction of RAS with PI3-kinase is required in established tumors. The need for RAS interaction with p110α in the maintenance of mutant Kras-driven lung tumors was explored using an inducible mouse model. In established tumors, removal of the ability of p110α to interact with RAS causes long-term tumor stasis and partial regression. This is a tumor cell-autonomous effect, which is improved significantly by combination with MEK inhibition. Total removal of p110α expression or activity has comparable effects, albeit with greater toxicities.

Coordinate Direct Input of Both KRAS and IGF1 Receptor to Activation of PI3 kinase in KRAS-Mutant Lung Cancer

UNLABELLED Using a panel of non-small cell lung cancer (NSCLC) lines, we show here that MAP-ERK kinase (MEK) and RAF inhibitors are selectively toxic for the KRAS-mutant genotype, whereas phosphoinositide 3-kinase (PI3K), AKT, and mTOR inhibitors are not. IGF1 receptor (IGF1R) tyrosine kinase inhibitors also show selectivity for KRAS-mutant lung cancer lines. Combinations of IGF1R and MEK inhibitors resulted in strengthened inhibition of KRAS-mutant lines and also showed improved effectiveness in autochthonous mouse models of Kras-induced NSCLC. PI3K pathway activity is dependent on basal IGF1R activity in KRAS-mutant, but not wild-type, lung cancer cell lines. KRAS is needed for both MEK and PI3K pathway activity in KRAS-mutant, but not wild-type, lung cancer cells, whereas acute activation of KRAS causes stimulation of PI3K dependent upon IGF1R kinase activity. Coordinate direct input of both KRAS and IGF1R is thus required to activate PI3K in KRAS-mutant lung cancer cells. SIGNIFICANCE It has not yet been possible to target RAS proteins directly, so combined targeting of effect or pathways acting downstream of RAS, including RAF/MEK and PI3K/AKT, has been the most favored approach to the treatment of RAS -mutant cancers. This work sheds light on the ability of RASto activate PI3K through direct interaction, indicating that input is also required from a receptor tyrosinekinase, IGF1R in the case of KRAS -mutant lung cancer. This suggests potential novel combination therapeutic strategies for NSCLC.

RAS mutation status and bortezomib therapy for relapsed multiple myeloma.

A.M. Schut participated in the design of the present study, helped to coordinate the study, performed experiments, analysed and interpreted data and wrote the manuscript. A. Venemans-Jellema performed data analysis, interpreted data and revised the manuscript. J. C. M. Meijers supervised some of the experiments, interpreted data and revised the manuscript. S. Middeldorp and F.R. Rosendaal participated in the study design, interpreted data and revised the manuscript. P. G. de Groot and M. Roest supervised some of the experiments, interpreted data and revised the manuscript. T. Lisman participated in the design and supervision of the present study, interpreted data and wrote the report. S.C. Cannegieter participated in study design, supervised data analysis, interpreted data and revised the manuscript.

Retraction: HMGA2 functions as a competing endogenous RNA to promote lung cancer progression

This corrects the article DOI: 10.1038/nature12785

Abstract A26: GATA2 is necessary for oncogene-mutant non-small cell lung tumorigenesis

Non-small cell lung cancer (NSCLC) is the most common histological cancer type in terms of both prevalence and mortality worldwide. In NSCLC, KRAS and EGFR are the most frequently mutated oncogenes, constituting half of all patients. Thus, there is a profound need to develop therapies targeting these lesions. To address this need, we previously performed an RNA interference screen for factors required in KRAS mutant cells. Through this screen, we found KRAS mutant cells depend on the transcription factor GATA2. Subsequent analysis of a broad panel of NSCLC lines revealed GATA2 depletion suppressed mutant cell viability. We then explored the role of GATA2 in NSCLC cells with a variety of oncogene mutations. We observed lethality with GATA2 loss in mutant NSCLC cells, with no effect in wild type NSCLC cells. Moreover, transplanted mutant NSCLC cells depleted for GATA2 exhibited abrogated tumor growth, while wild type tumors were unaffected, suggesting GATA2 is needed in oncogene-mutant NSCLC. To elucidate the mechanism of GATA2 function in NSCLC gene expression, we performed gene expression analysis of mutant cells with GATA2 suppression. With GATA2 loss, we observed global downregulation of the proteasome and subsequent inhibition of proteasome activity. This inhibited proteasome function is biologically salient, as restoration of proteasome activity rescued growth in response to GATA2 depletion. We further explored the role of GATA2 in NSCLC by integrating the above gene expression data with global genome occupancy with chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq). Through these approaches, GATA2 loss suppressed expression of the IL-1 signaling pathway in mutant cells, down-regulating pathway flux. Consequently, restored IL-1 signaling reestablished growth in response to GATA2 depletion. Moreover, GATA2 occupancy was enriched in Rho signaling pathway components, functionally inactivating Rho signaling. Accordingly, activated Rho signaling rescued viability in response to GATA2 loss. Analyzing the DNA motifs from our GATA2 ChIP of mutant NSCLC cells, we observed enrichment of the STAT5 response element at Rho target genes. We subsequently isolated a GATA2-STAT5 complex in mutant NSCLC cells. Overall, our integrative genomic analyses have revealed a transcriptional network governed by GATA2 in oncogene-driven NSCLC. To examine the effect of GATA2 loss in genetically engineered mouse models (GEMMs) of NSCLC, we combined a conditional allele of Gata2 with Cre-inducible oncogenic Kras, where Gata2 deletion suppressed tumor growth compared to wild type animals. We then examined the role of whole-body loss of GATA2 in established lung tumors via systemic deletion of Gata2. In this system, GATA2 depletion caused complete regression of established lesions. We then explored whether inhibition of GATA2-regulated pathways with clinically approved compounds would recapitulate the effects of GATA2 loss in NSCLC GEMMs. To do so, we combined bortezomib, an inhibitor of the proteasome, and fasudil, an inhibitor of Rho signaling, in the Kras lung tumor model. Treatment of the Kras-mutant GEMM with these compounds caused a near-complete clearance of tumors. These combined genetic and therapeutic approaches reveal GATA2 is required for autocthonous oncogene-driven tumorigenesis and that combined inhibition of GATA2-controlled pathways with licensed agents suppresses NSCLC growth. In sum, these results demonstrate GATA2 is necessary for oncogene-mutant NSCLC cell survival. Most strikingly, these findings suggest that the functional pleiotropy of GATA2, not a traditional druggable target, represents a network of druggable pathways for therapeutic exploitation.

Abstract PR14: Targeting the GATA2 transcriptional network in K-Ras driven lung adenocarcinoma

Non-small cell lung cancer (NSCLC) is the most prevalent histological cancer subtype in terms of both incidence and mortality, with nearly 1.4 million deaths per year worldwide. Nearly one-third of all NSCLCs have activating mutations in the KRAS oncogene. In spite of thirty years9 knowledge of KRAS mutations in lung cancer, it has proven therapeutically intractable, leaving KRAS mutant NSCLC to serve as a marker of poor prognosis. Thus, there is a substantial unmet clinical need to find treatment options for this subset of lung cancers. By RNA interference screening, we previously discovered a transcription factor, GATA2, is necessary for the survival of KRAS mutant lung cancer cells, but not wild type lung cancer cells. Through an integrative genomic analysis of gene expression and chromatin occupancy, we discovered a GATA2-regulated transcriptional network consisting of the proteasome, IL-1 and Rho pathways. Functional analyses revealed that each of these pathways, though not individually necessary, is partially sufficient to restore viability in response to GATA2 depletion. Using two different genetically engineered mouse models (GEMMs), we found Gata2 is necessary for oncogenic Kras driven lung tumourigenesis. In addition, systemic Gata2 loss, while well tolerated, significantly extended survival in a Kras mutant NSCLC GEMM. Intriguingly, we found that combined treatment of Kras driven mouse models with clinically available inhibitors of the proteasome (bortezomib) and Rho kinase (fasudil) induced tumour regression. We have now begun exploring and extending these initial therapeutic strategies to the Kras; Trp53 (KP) mouse model. Upon delivery of Cre recombinase to the lungs, KP mice undergo progression from low-grade adenomas to high-grade, invasive metastatic lung adenocarcinoma, more faithfully recapitulating the human disease. We have assessed our GATA2 network therapies through in vivo imaging of incipient lesions by micro-computed tomography, treatment cycles and subsequent re-imaging to determine efficacy. Using this approach, we observed substantial response when combining treatment with bortezomib and fasudil in the KP model. Moreover, introduction of a next-generation inhibitor of Rho kinase (AT13148), an agent in early-stage clinical development, combines with bortezomib to cause significant regression of lung adenocarcinomas in vivo . Taken together, we have elucidated a gene expression network necessary for Ras-driven lung cancer and have devised novel approaches to suppress this network with therapeutically tractable agents. This abstract is also presented as Poster A14. Citation Format: Madhu S. Kumar, Julian Downward. Targeting the GATA2 transcriptional network in K-Ras driven lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR14.

Abstract B221: Hmga2 furthers lung adenocarcinoma progression through function as a competing endogenous RNA.

Lung adenocarcinoma is the most widespread cancer type worldwide. As the majority of patients are diagnosed with metastatic disease, it is vital to understand that biological basis of adenocarcinoma progression. Hmga2 is among the most over-expressed genes in lung adenocarcinoma, and we have previously shown it is necessary for lung cancer progression in vivo. However, the mechanism of Hmga2-mediated cancer progression is unclear. Here we demonstrate Hmga2 promotes oncogenic transformation by functioning as a competing endogenous RNA (ceRNA) for the let-7 family of microRNAs (miRNAs). First, Hmga2 can promote lung cancer cell transformation independent of its protein-coding function but dependent upon let-7 sites in its 3’ untranslated region. These effects are observed both in vitro and in vivo, where Hmga2 ceRNA activity promotes lung cancer growth and dissemination, impairing survival. Combined analyses of miRNA target prediction and cell line gene expression data suggested the TGF-β co-receptor Tgfbr3 lies downstream of the Hmga2 ceRNA. Follow-up studies demonstrated Hmga2 specifically regulated Tgfbr3 expression through differential recruitment of the miRNA regulatory machinery. This regulation is functionally relevant, as both Tgfbr3 in particular and TGF-β in general are necessary for Hmga2 to promote transformation. To assess the clinical relevance of these experimental findings, we assessed gene expression data from two extensive lung cancer patient gene expression datasets: the Cancer Genome Atlas and the Director9s Challenge. Analysis of this gene expression data revealed that HMGA2 and TGFBR3 are coordinately and reciprocally regulated in patients, a necessary prediction of HMGA2 ceRNA function. In total, these results indicate the Hmga2 oncogene functions as both a protein-coding gene and a ceRNA. Such multi-level regulation of gene expression constitutes a novel paradigm by which genes alter cancer development and progression through combined function as both coding proteins and non-coding RNAs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B221. Citation Format: Madhu S. Kumar, Elena Armenteros-Monterroso, Philip East, Probir Chakravorty, Nik Matthews, Monte M. Winslow, Julian Downward. Hmga2 furthers lung adenocarcinoma progression through function as a competing endogenous RNA. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B221.

Abstract PR-5: GATA2 is required for oncogene-driven non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most diagnosed and lethal histological type of cancer worldwide. Among NSCLC patients, the most frequently mutated oncogenes are KRAS and EGFR, constituting 40 percent of patients. Thus, there is a need to develop therapies to target these tumors. We performed a RNA interference screen for factors required in KRAS mutant cells and found mutant cells depend on the transcription factor GATA2. Analysis of 26 NSCLC lines revealed GATA2 loss specifically reduces KRAS mutant cell viability. We explored these effects in NSCLC cells mutant for KRAS or EGFR. GATA2 knockdown reduces the viability of NSCLC cells with activating mutations in either KRAS or EGFR, with wild type NSCLC cells growing comparably well. Furthermore, injection of mutant NSCLC cells depleted for GATA2 failed to form tumors, while wild type tumors were unaffected by GATA2 loss. This suggests GATA2 is required specifically in oncogene-driven NSCLC. To determine the effect of GATA2 on gene expression, we performed transcript profiling of mutant cells with and without GATA2 loss. In response to GATA2 loss in NSCLC cells, we observed suppression of the proteasome. GATA2 loss inhibited activity of the proteasome and restoration of proteasome activity rescued growth in response to GATA2 depletion. Furthermore, GATA2 loss suppressed IL-1 and NF-κB signaling in mutant NSCLC cells. Restoring IL-1 signaling reestablished both NF-κB activity and growth in response to GATA2 knockdown. We further explored GATA2 genome occupancy and gene expression through GATA2 ChIP-seq in NSCLC cells. In mutant NSCLC cells, GATA2 occupancy was enriched in Rho signaling pathway components. Further, we found GATA2 loss suppressed Rho signaling and activation of Rho signaling rescued viability in response to GATA2 loss. Analyzing the DNA motifs from our GATA2 ChIP of mutant NSCLC cells, we observed enrichment of the STAT5 response element. We subsequently isolated a GATA2-STAT5 complex in mutant NSCLC cells; importantly, this complex occupies Rho pathway target genes. Overall, our ChIP-seq suggests GATA2, via STAT5 binding, is recruited to Rho pathway target genes to control Rho signaling. To examine GATA2 loss in an autochthonous model of NSCLC, we combined conditional Gata2 with oncogenic Kras to generate compound mutant animals. Gata2 deletion suppressed tumor growth compared to wild type animals. We further tested the role for GATA2 in tumor maintenance via systemic deletion of Gata2 in established Kras-mutant lung tumors, which caused complete regression of established lesions. Beyond this, we combined bortezomib, an inhibitor of the proteasome, and fasudil, an inhibitor of Rho signaling, to see if suppression of GATA2-regulated pathways recapitulates GATA2 loss in the Kras lung tumor model. Treatment of Kras mutant mice with these compounds caused a near-complete clearance of tumors. These studies suggest GATA2 is necessary in the Kras mutant tumor model and combined inhibition of GATA2-controlled pathways suppresses lung tumor growth. Taken together, these findings suggest GATA2 is required for the survival of oncogene-mutant NSCLC cells. Moreover, these results suggest the functional pleiotropy of GATA2, not a traditional druggable target, presents a network of druggable pathways for therapeutic exploitation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-5.