For almost 30 years, the protein KRAS has been the ‘undruggable target’ of cancer research. Gene mutations in KRAS are responsible for 33% of all human cancers, including pancreatic, lung, and colon cancer.
A new molecule identified by Dr Bhairavi Tolani, Assistant Professor at the University of California, San Francisco, upends conventional concepts of directly targeting the protein as a treatment strategy. Instead she suggests an alternative avenue for treatment of these deadliest cancers.
Read her original research in Nature Biotechnology here: https://doi.org/10.1038/s41587-022-01386-z
Image source: Tilia Lucida/ Shutterstock
Transcript:
Hello and welcome to Research Pod! Thank you for listening and joining us today.
In this episode, we will discuss the unexpected discovery of a new molecule that could become a treatment for some of the deadliest cancers, identified by Dr. Bhairavi Tolani, an assistant professor at the University of California, San Francisco. Tolani upends conventional concepts of directly targeting the protein KRAS as a treatment strategy for KRAS-driven cancers; instead, as published in her article in Nature Biotechnology, she suggests an alternative avenue for treatment.
For almost 30 years, the protein KRAS has been the “undruggable target” of scientists in academic laboratories, the biopharma industry and government institutions alike. Due to the failure of efforts at direct pharmacological inhibition of Ras mutants. But this target is worth pursuing as it’s responsible for 33% of all human cancers. Specifically, gene mutations in KRAS are responsible for the deadliest cancers, including pancreatic, lung, and colon cancer.
In 2013, enthusiasm was reignited, as a direct KRAS-G12C inhibitor molecule was made by Dr. Kevan Shokat, another professor at UCSF. This molecule targets a subset of KRAS proteins, but only when there is mutation or change to the amino acid Cysteine at position 12 – thus the term KRAS-“G12C”.
Shokat’s molecule was developed into a drug called Lumakras by the biotechnology company, Amgen. It secured approval from the US Federal Drug Administration in 2021 allowing its’ use in patients who have KRASG12C-mutated cancers . But the mutation KRAS-G12C accounts for only 11% of all KRAS cancers leaving the remaining KRAS mutations, open for targeting. More importantly, many patients who were given Lumakras have now stopped responding to it, as their tumours have developed resistance to the drug. Making the clinical need for alternatives and the next generation of drugs is all the more urgent.
A team of scientists led by Tolani across 7 universities – including UCSF, Harvard, MIT and Stanford -discovered an alternative route to direct pharmacological inhibition of KRAS using 249C, a small molecule inhibitor of a protein called “V-ATPase.” The team provided the conceptual connection between targeting V-ATPase and sensitivity to various KRAS mutation types; but also presented a new molecule,249C, which targets cancers with specific KRAS-G13D and KRAS-G12V mutations in a new and indirect way.
249C is highly potent at killing cancers with various RAS mutations not just those with single KRAS mutations. In their latest paper, the team describe the discovery, target identification, and mechanism by which 249C works. 249C being most active for KRAS-G13D and KRAS-G12V cancers.
The origins of 249C’s development begins Six months prior to Tolani’s arrival at UCSF in 2014, when a staff chemist created molecules targeting a protein active in lung cancer. Efforts to block such proteins based on their 3-dimensional structures, via molecules like 249C, is a common drug discovery approach. Over a period of 1.5 years, the chemist made approximately 300 molecules and Tolani iteratively tested their ability to kill cancer cells.
Tolani discovered 249C was the most potent and suitable molecule from ~300 originally designed candidates. However, these molecules, many of which are highly potent, had been structurally tweaked extensively. Tolani wondered if 249C, potent as it was, could attach to other targets within cells. Which could make 249C’s mechanism of action different than what was originally intended.
Dissecting the mechanism by which a molecule like 249C exerts its killing activity and identifying the exact target of 249C from scratch – when there are over 20,000 proteins present within cells – is no small feat. But it is one of the most significant scientific problems to solve.
In her quest to solve this problem and pinpoint the exact cellular target of 249C, Tolani teamed up with Dr. Jonathan Weissman at MIT and Dr. Marco Jost at Harvard). Both are experts of CRISPR interference and chemical–genetic screening, which has emerged as a robust strategy for identification of small molecule targets. Weissman along with Nobel laureate, Dr. Jennifer Doudna, recently developed revolutionary CRISPR-based tools which can be used to determine the cellular target of small molecules such as 249C.
From their CRISPR experiment, the team found that several parts of the V-ATPase protein were genetically effected by 249C treatment. Reassuringly, three independent experiments, CRISPR-based chemical–genetics, chemo-proteomics, and comparative profiling, all implicated V-ATPase as the cellular target of 249C.
To validate and confirm these findings, the team joined forces with V-ATPase expert, Dr. Rubinstein and his lab, at the University of Toronto. Using biochemical and biophysical techniques, the team found that 249C attached to V-ATPase quite strongly and inhibited its biochemical activity. Upon attaching to V-ATPase, 249C prevents crucial cellular processes called autophagy and macropinocytosis (MP) which several KRAS cancers depend on for survival.
Unexpectedly, Tolani discovered that the potency of 249C differed based on the Ras mutation present in cells. She discovered a pattern by testing cells from the National Cancer Institute, where each cell population contains a unique KRAS mutation. She found that 249C had the highest potency for mutations KRAS-G13D and KRAS-G12V; both in cells grown in the lab and in living mice. Highlighting a mutation-specific dependence on V-ATPase and the process of macropinocytosis, which can be exploited by treatment with 249C.
Given its promising activity to shrink tumours in mice, low toxicity, and favourable pharmacokinetic characteristics; 249C was an ideal candidate for the development of a drug to treat KRAS-G13D and KRAS-G12V driven cancers. The G13D and G12V mutations account for 13% and 24% of KRAS mutations across all human cancers, respectively. Giving 249C the potential to treat ~37% of all KRAS cancers.
Coincidentally, 3 studies have recently been published centring on the Ras-V-ATPase autophagy / Macropinocytosis connection. NYU’s Dr. Bar-Sagi’s lab reported that V-ATPase controls macropinocytosis prompted by KRAS in a 2019 Nature article. UNC Chapel Hill’s Drs. Der and Cox showed that mutant KRAS-G12R is impaired in macropinocytosis in 2020.),Finally, UCSF’s Dr. Frank McCormick speculated that lysosomal inhibitor molecules could inhibit Ras tumours in 2021.
In fact, 249C *is* a lysosomal inhibitor molecule. 249C (also called RSC 1255) has already been in clinical trials for 2 years. Tolani is an inventor on the UCSF patent covering 249C and this family of molecules; which has been licensed to a biotechnology start-up. In 2020, the start-up launched a Phase I clinical trial for the following indications: Ras mutations, lung cancer, colon cancer, pancreatic cancer, and glioblastoma across six different sites in the U.S, and recruitment is ongoing.
For over 30 years KRAS has been the nemesis of cancer researchers across the globe, as it is responsible for one-third of all human cancers. Even though Shokat’s and Amgen’s LUMAKRAS molecule is exciting, KRAS-G12C mutations are present in ~11% of all KRAS cancers; and tumours treated with LUMAKRAS have become resistant. Thus, the clinical need for alternatives is urgent.
Tolani and team discovered and patented the highly potent molecule 249C which is active against a number of Ras mutations but shows strongest activity in KRAS-G13D and KRAS-G12V which account for ~37% of all KRAS cancers.
Dr. Tolani believes the clinical impact of this work could translate into improved treatment outcomes for cancer patients with aggressive KRAS mutations – particularly those with KRASG13D and G12V – by shrinking their tumours and increasing their survival. However, she cautions against the drug candidate being dubbed as a ‘silver bullet’ cure, and urges us to not jump to any conclusions until the Phase I clinical data are analysed for safety in the next 2 years.
That’s all for this episode – thanks for listening and be sure to check out the Nature Biotechnology paper from Dr Tolani, linked to in the show notes for this episode, for more details. And as ever, stay subscribed to Research Pod for more of the latest science.
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