Diving into the dark kinome to identify synthetic lethal RAS therapeutic targets

From the Kemp Lab, Human Biology Division

When it comes to finding therapeutic cancer targets, “mutated cancer genes are just the tip of the iceberg, but functional genomics can help uncover the much larger, submerged portion of the iceberg,” explains Dr. Christopher Kemp, Professor in the Fred Hutch Human Biology and Public Health Sciences Divisions. There is an emphasis put on finding therapeutic treatments that target cancer-driving mutations, but in reality, “cancer cells have lots of vulnerabilities beyond what people typically look for,” adds Dr. Kemp. The Kemp lab focuses on uncovering these vulnerabilities, or this submerged portion of the iceberg, with an emphasis on investigating “the dark kinome”. The kinome refers to hundreds of kinase enzymes that play critical roles in signaling cascades and lead to activation or inhibition of their target proteins, however the roles of many of these kinases remain poorly characterized. Cancer-driving mutations can rewire normal cellular functioning and lead to dependencies on specific kinases to help fuel their unregulated growth. These kinases create cancer cell-specific vulnerabilities, as inhibiting one of these synthetic lethal kinases would negatively affect only the growth of cancer cells which have become dependent on them, while healthy cells remain largely unaffected.. In their recently published Oncogene paper, the Kemp Lab aimed to exploit the cancer kinome and find the targetable weak spots of cancer cells by identifying kinases necessary for cell growth in RAS-driven squamous cell carcinoma (SCC).

Mutations in RAS family members Hras and Kras occur frequently across a number of human cancers including SCC. However, despite decades of research, these cancers are still largely considered undruggable as most identified putative targets have failed to advance clinically. Rather than target these mutated genes themselves, Moser et al. sought to identify kinases in downstream oncogenic RAS signaling pathways that mutant RAS cells rely on for growth and survival. To do so, the researchers utilized a well-established, chemically-induced (through treatment with chemical mutagen DMBA and tumor promoter TPA) SCC mouse model that results in 80-90% of skin tumors that harbor a mutation in Hras and to a lesser extent, Kras. Since in human cancers RAS genes are commonly co-mutated with DNA damage response genes, the researchers induced this RAS-driven SCC model in various mouse genetic backgrounds, some of which had an additional mutation in a DNA damage response gene such as Trp53 or Atm. The researchers then derived seven cell lines from tumors with various SCC genetic backgrounds to use for their dark kinome investigation. Taking a large-scale, unbiased approach, the Kemp team performed an arrayed siRNA screen in these seven derived SCC cell lines to knock down roughly 600 kinase genes individually, rather than a pooled format. This design enabled the researchers to identify which kinases RAS mutant cancer cell lines commonly depended on for their growth, as well as investigate how these dependencies change when RAS mutant cells harbor an additional mutation in a DNA damage response pathway gene. While it requires a lot more work, asking how knocking down 600 kinases individually affects each of these different cell lines offers the advantage of a more reproducible and sensitive approach compared to knocking them all down in a pooled fashion, which can often miss subtle phenotypes. Screening ~600 kinases in 7 different cell lines, in triplicate, however, yields a lot of data. Kemp notes that one of the biggest challenges of this project was trying to figure out how to then interpret all of that data.

Synthetic lethal screen strategy to identify kinase therapeutic targets of RAS-driven SCC.
Synthetic lethal screen strategy to identify kinase therapeutic targets of RAS-driven SCC. Image taken from original article

The authors took various approaches to identify important and biologically meaningful putative RAS therapeutic targets including screening nearly 100 small molecule kinase inhibitors that are FDA approved or in clinical development for use in cancer therapy. Collectively, the Kemp Lab’s combined siRNA and drug profiling identified both previously reported and novel kinase targets that function within and beyond RAS effector pathways. These targets include kinases involved in PI3K/mTOR signaling, cell cycle and epigenetic regulators, Map kinases, and DNA damage response. Also among these hits were a set of lipid kinases that exhibited differences in their effect on different Hras mutant cell lines, revealing that RAS dependencies can exhibit incomplete penetrance, that is synthetic lethality is dependent on the precise genetic backgrounds. Another exciting target discovered was the mitotic kinase Nek4 which exhibited robust dependency in both mouse and human SCC cell lines, thus demonstrating its therapeutic potential.

Since the functional profiling performed in this study used cells derived from a chemically-induced SCC mouse model with roughly 100 years of rich experimental history, it meant the findings could be “anchored in decades of research, so we know what we are finding has physiological relevance,” Kemp states. Furthermore, this study was the first to perform a comprehensive profiling of this long-standing model, as it systematically investigated all possible downstream kinase signaling cascades to probe for previously unknown or underappreciated vulnerabilities, thus greatly accelerating what we understand about this common RAS-driven cancer model. This work further highlights the power of functional genetics and provides hope that we will eventually be able to efficiently target these currently “undruggable” cancers. Dr. Kemp notes that future studies aim to “develop inhibitors to the kinase targets identified in this work and test them in preclinical models.” He concludes by acknowledging that the early stages of this work were supported by Dr. Lee Hartwell- the previous director and president of Fred Hutch and Nobel Laureate who was one of the first researchers to propose the idea of identifying novel cancer targets through synthetic lethality. Furthermore, key contributors included the late Dr. Eddie Méndez, a Fred Hutch physician-scientist dedicated to improving the lives of patients with head and neck cancer, and Dr. Carla Grandori from SEngine Precision Medicine- a company co-founded by Kemp, Grandori, and Méndez.

This work was supported by the National Institutes of Health, the National Cancer Institute and the American Cancer Society.

Fred Hutch/UW Cancer Consortium member Christopher Kemp contributed to this work.

Moser R, Gurley KE, Nikolova O, Qin G, Joshi R, Mendez E, Shmulevich I, Ashley A, Grandori C, Kemp CJ. Synthetic lethal kinases in Ras/p53 mutant squamous cell carcinoma. Oncogene. 2022 Jun;41(24):3355-3369. doi: 10.1038/s41388-022-02330-w. Epub 2022 May 10. PMID: 35538224.