Opening a new GATAway for treating KRAS-driven lung tumors

Abstract

In a recent issue of Cell, Downward and colleagues have uncovered a synthetic lethal interaction between oncogenic KRAS and the transcription factor GATA2 in NSCLC. Pharmacological inhibition of GATA2-mediated pathways with bortezomib (proteasome) and fasudil (RHO/ROCK) results in dramatic tumor inhibition. These observations unveil new armamentaria to fight this deadly disease.

Non-small cell lung carcinoma (NSCLC) has one of the highest incidence and lowest survival rates, a combination that makes this tumor type one of the deadliest human cancers. At least a quarter of NSCLC express a mutant KRAS allele that encodes a constitutively active small G-protein known to signal through a series of kinases. While these kinases are in principle amenable to pharmacological intervention, a selective treatment for KRAS mutant NSCLC is not yet available in the clinic.

During the last decade, there have been significant efforts directed to reproduce the natural history of NSCLC in genetically engineered mouse (GEM) models (Heyer et al., 2010). Recently, these models have been utilized to evaluate potential therapeutic targets. Some of the validated targets include well-known downstream elements of KRAS signaling such as components of the mitogenic RAF/MEK/ERK cascade and the PI3K/AKT survival pathway, most of which are druggable kinases (Gupta et al., 2007; Engelman et al., 2008; Blasco et al., 2011) (Figure 1A). However, these studies cannot be extrapolated to the clinic because target ablation occurred during tumor initiation rather than during tumor progression. Moreover, these Kras oncogene-driven GEM models retained the full component of tumor suppressors and, hence, do not develop metastatic tumors.

Figure 1. Preclinical studies to validate therapeutic targets in KRAS-driven NSCLC.

(A) Schematic diagram of putative therapeutic targets validated by genetic approaches in GEM models of NSCLCs driven by resident oncogenic Kras. (Left) GEM models driven by conditional mutant Kras alleles develop adenomas and non-metastatic adenocarcinomas (in blue). (Right) GEM models that also carry mutated or null Tp53 or have Tp53 knockdown develop more aggressive adenocarcinomas (in red) that frequently metastasize to distant organs. (B) RNAi screens aimed at identifying human genes capable of inducing synthetic lethality in combination with KRAS oncogenes. (C) Pharmacologic approaches using selective targeted inhibitors in the above Kras-driven GEM models. Original references for these GEM models have been omitted due to space limitations.

Other studies have attempted to validate pathways less directly linked to KRAS signaling. In one study, elimination of Cdk4, but not the other interphase Cdks, elicited a rapid senescence response that resulted in partial tumor regression, an observation validated with clinically available CDK4 inhibitors (Puyol et al., 2010) (Figure 1A). Other studies have examined the therapeutic consequences of inhibiting the NF-kB pathway in GEM models carrying Tp53 null alleles or expressing reduced levels of p53 (Meylan et al., 2009; Xia et al., 2012). When challenged with an IkB super-represor or when IKKβ expression was knocked down, tumors regressed, althoguh they were not completely eliminated (Figure 1). These observations suggest that blocking NF-kB-driven survival pathways might serve as a therapeutic strategy to thwart KRAS-driven tumor progression.

Other investigators have expanded the repertoire of KRAS targets by searching for synthetic lethal genes using RNA interference (RNAi) libraries. Screens of human NSCLC cell lines carrying either wild-type or mutant KRAS oncogenes have identified targets linked to the cell cycle such as PLK1, a kinase involved in mitotic progression and to the NF-kB pathway such as TKB1, a non-canonical IkB kinase (Barbie et al., 2009; Luo et al., 2009) (Figure 1B). In vivo validation of these synthetic lethal genes will establish their therapeutic value in KRAS driven lung tumors.

In a recent issue of Cell the Downward laboratory has gone all the way from identifying GATA2 as a novel synthetic lethal gene to validating it using Kras-driven GEM models and finally, to demonstrating its therapeutic potential by using surrogate drugs already approved for clinical use (Kumar et al., 2012) (Figure 1). Kumar et al., screened human NSCLC cell lines carrying either wild-type or mutant KRAS genes with an RNAi library against 7,000 human genes. Using cell viability as the biological read-out, they identified the transcription factor GATA2 essential for the proliferation of cell lines carrying oncogenic KRAS. GATA2 knockdown also reduced the viability of cells lines carrying mutated loci functionally related to KRAS such as NRAS, NF1, EML4-ALK and EGFR. Interestingly, GATA2 is an unlikely actor in lung cancer since its fucntion has so far only been linked to the hematopoietic system.

Gene set enrichment analysis (GSEA) revealed that reduction of GATA2-mediated transcription in lung tumor cells affected several pathways, suggesting that a pleiotropic effect is required for the observed synthetic lethality. Knockdown of GATA2 affected proteosomal activity in lung tumor cells, an observation reminiscent of that obtained in previous screens (Barbie et al., 2009; Luo et al., 2009). This effect appears to be independent of the presence of oncogenic KRAS and is not sufficient to induce loss of cell viability. GATA2 depletion also led to transcriptional repression of IL-1 and NFκB signaling pathways. In this case, some of the observed effects, mainly expression of TRAF6, were specific for KRAS mutant cells. Yet direct inhibition of either IL-1 signaling by its antagonist IL-1a or of NF-κB signaling by TRAF6 knockdown did not result in loss of cell viability, suggesting that inhibition of either pathway alone is not sufficient to phenocopy loss of GATA2 expression. Finally, Kumar et al., identified that some genes involved in RHO-related signaling pathways were occupied by GATA2 in KRAS mutant but not wild-type tumor cells. Not surprisingly, GATA2 knock down in KRAS mutant cells resulted in a striking reduction of active GTP-bound RHO proteins, including the downstream ROCK kinase. Restoration of RHO activity by expression of constitutively active RHO proteins or by a ROCK-ER fusion kinase, did not rescue cell viability in the absence of GATA2, again suggesting that blocking RHO signaling is not sufficient to mimic loss of GATA2 activity. Intriguingly, KRAS oncogene knockdown, while affecting cell viability of KRAS mutant cells to a similar extent as GATA2 knockdown, had no effect on the proteasome pathway, NF-κB activity or RHO signaling. These observations suggest that GATA2 is not a downstream component of the KRAS oncogenic signaling pathway.

Conditional ablation of Gata2 alleles in a Kras-driven GEM model of NSCLC prevented tumor initiation. More importantly, systemic elimination of Gata2 in mice already presenting Kras-driven lung tumors resulted in substantial tumor regression without major side effects suggesting that blocking GATA2 activity could have therapeutic value and might be well tolerated in patients. Whether inhibition of GATA2 may result in defects in their immune system, a phenotype that might not be obvious in the protected environment of an animal facility, remains to be determined.

Finally, Kumar and co-workers combined available inhibitors selective for two of the pathways regulated by GATA2 to treat mice with Kras-driven NSCLCs. The chosen inhibitors bortezomib, a proteasome inhibitor, and fasudil, a RHO/ROCK inhibitor, have already been approved for use in human patients. When combined, these inhibitors induced almost complete regression of well-established lung tumors in the Kras-driven GEM model (Figure 1C). These observations are reminiscent of a previous report in which similar tumors also regressed upon treatment with a combination of MEK and PI3K inhibitors (Engelman et al., 2008). However, MEK inhibitors have not been approved by the FDA due to undesired toxicities and PI3K inhibitors have thus far shown rather limited anti-tumor activity.

Are we on the verge of a major breakthrough in the treatment of KRAS-induced NSCLC?. Possibly, however, we should consider the data of Kumar et al., (2012) as an exciting but early step in the long process of drug discovery. The GEM model used in this study retains wild-type Tp53 suggesting that the tumors successfully treated with bortezomib and fasudil might not be as aggressive as those in most NSCLC patients. Moreover, the in vivo data is still preliminary and other potential roadblocks such as drug resistance have not been examined. In spite of these caveats, the results of Kumar et al. represent a very important advance in the long-standing fight to conquer lung cancer. Undoubtedly, they have opened an important “GATAway” towards this challenging goal.