Adventures with the MAPK pathway

A collection of ongoing examples of curiosity-driven research continuously supporting clinical development

This article has been inspired by Prof Bernards’ research presented in the EACR’s #KeepResearchCurious webinar Chance favours the prepared mind, my adventures with the MAP kinase pathway’ and Professor Mariano Barbacid’s lecture for the 2024 Mike Price Gold Medal award.

1. Half a century’s worth of research: from viral oncogenes to FDA approved small molecule inhibitors of Ras

The MAPK pathway is a key signaling pathway in mammalian cells. Also known as RAS-RAF-MEK-ERK pathway, it is a complex, interconnected signaling cascade that integrates, amplifies, and transduces extracellular signals into intracellular responses and thus sustains many essential cellular processes like proliferation, differentiation, development, transformation, and apoptosis. Reflective of its complexity and importance, alterations of this pathway can lead to various diseases, including several forms of cancer [1].

The first connection between cancer development and the MAPK pathway dates from 1982, when the first human oncogene was isolated by Prof Mike Wigler [2], Prof Robert Weinberg  [3], and Prof Mariano Barbacid [4] the latter two having identified this oncogene as a transforming allele of H-RAS, the human homologue of the retroviral v-Ha-Ras oncogene [5, 6]. This came about a decade after the physical identification of the first oncogene: src by Duesberg and Vogt in 1970, then believed to be a retroviral gene [7] and 6 years after Mike Bishop’s and Harold Varmus’ Nobel prize discovery that src was in fact a mutated allele of a cellular gene picked up by retroviral recombination [8]. These discoveries laid the groundwork for the establishment of the molecular bases of oncology.

Later the same year, Prof Geoffrey Cooper’s lab identified K-Ras in mouse fibroblast cells transformed with DNA from human lung cancer cells [9]. Shortly after, KRAS was identified in several solid human tumours [10] and a clear connection between KRAS and human tumours was established when the Barbacid lab showed that mutated KRAS is present in tumoral, but not healthy tissue from lung cancer patients [11]. However, it took more than 30 years until a KRAS selective inhibitor was developed [12].

Since 1982 the MAPK pathway and its crosstalk with other signaling pathways has been studied in great detail in homeostasis and disease [1, 13-16]. RAS mutations, specifically in the KRAS allele, are the most frequent oncogenic event in human cancers being found in a variety of solid tumours such as pancreatic, colorectal, lung adenocarcinomas and urogenital cancers [17]. The MAPK pathway has therefore understandably received increasing interest from the pharmaceutical industry over the past couple of decades as proven by the high number of drugs developed to inhibit this pathway (Fig 1). Starting from the cell membrane, at the level of the receptor tyrosine kinases, and going down the enzymatic cascade of the pathway, we can see that many potent inhibitors, antibodies, and small molecules have been developed, many of which have been approved by FDA and in clinical use today. Starting at the level of the cell membrane with RTK inhibitors like Osimertinib, Crizotinib, Axitinib, Lenvatinib just to name a few of those approved by FDA and continuing with the agents that transmit the signal to RAS, such as SHP2 inhibitors and SOS1 inhibitors before moving downstream to the latest generation RAS inhibitors like KRASG12C inhibitors such as Sotorasib and Adagrasib; Pan-RAS inhibitors; BRAF inhibitors like Dabrafenib/ Encorafenib; MEK inhibitors like Trametinib, Binimetinib, Mirdametinib and lastly ERK inhibitors (only FDA approved inhibitors were mentioned by their commercial name. All other types of inhibitors mentioned are still in various stages of research) [13].

Despite the high number of potent and selective drugs for this pathway, RAS driven tumours are often still not curable. This is in part due to the heterogeneity in treatment response between different tumour types, and in part because even tumours that are initially responsive to the treatment, quickly develop resistance to therapy. There is therefore a clear need to gain additional biological insights into the molecular mechanisms of signal transduction through the MAPK pathway and its crosstalk with other signaling pathways.

2. Targeting the “undruggable” a continuous battle to understand and ‘outsmart’ cancer cells

2.1. Prof. Bernards’ and Prof. Bardelli’s adventures with the MAPK pathway for the improvement of lung and colorectal cancer treatments

2.1.1. BRAF inhibitors: adapting melanoma treatments to fight colorectal cancer

One of the first inhibitors of the MAPK pathway to be successfully used in patients was the selective BRAF inhibitors for the treatment of BRAF mutant melanoma, such as Vemurafenib [18, 19]. Interestingly, this approach proved largely ineffective in BRAF mutant colorectal cancer patients fostering the same mutation in BRAF as the melanoma patients [20]. This caught the attention of EACR Board members Prof. Rene Bernards from NKI and Prof. Alberto Bardelli from IFOM who were puzzled as to why BRAF inhibition is sufficient to get at least initially a favorable response in melanoma but not in colorectal cancer.

Prof Bernards and Prof Bardelli started looking for a synthetic lethality approach that could improve treatment for colorectal cancer. In this approach inhibition of two proteins by administrating two different drugs would be able to kill tumour cells, whereas inhibition of each individual protein would not kill the cancer cells. Using the expertise from his lab, Prof Bernards was the first one to publish, in 2002, a vector capable of silencing mammalian genes long-term [21] and having pioneered barcoding [22], Prof Bernards started a loss of function screen looking at all kinases from the human genome in a BRAF colorectal cancer cell line that does not respond to Vemurafenib. Following the knockdown of the genes of interest, cells were treated with Vemurafenib in an effort to identify any inhibited kinase that would be toxic only in combination with Vemurafenib, but not by themselves. This experiment revealed EGFR as a promising target for a combinatorial treatment [23]. In our #KeepResearchCurious webinar, Prof Bernards notes “EGFR was, of course, very favorable from a clinical translational perspective, because there were already Antibodies like Cetuximab and Panitumumab used in the clinic and also small molecules, EGFR inhibitors like Gefitinib”. When moving the experiment in mouse models, the combined inhibition of BRAF and EGFR showed the strong toxicity suggested by the cell culture experiments, giving researchers “complete tumor control” [23].

These favourable results warrant translating this research into the clinic (Fig 2). Prof Bernards recalls “since I work at the Netherlands Cancer Institute, which is affiliated with a specialized cancer hospital, we were very fortunate that soon after the publication of this result of synthetic lethality between BRAF and EGFR, we were able to initiate a Phase 1B trial where we exposed BRAF mutant colorectal cancer patients to Encorafenib, a BRAF inhibitor, and Cetuximab, the antibody inhibitor, inhibiting, EGFR. And we saw indeed the first signs of clinical activity in those patients, which resulted in a BEACON study, a phase three trial that was published in the New England Journal in 2019. And that resulted in an FDA approval and EMA approval for the combination of oncorafenib and cetuximab” [24, 25].

2.1.2. An attempt to increase the efficacy of MEK inhibitors: efficacy vs toxicity, a balancing act

Encouraged by these favourable results, Prof Bernards and Prof Bardelli alongside their teams and collaborators were hoping that EGFR inhibitors would be the answer to a similar dilemma that arose in the MAPK cancer research field: the lack of therapeutic benefit following inhibition of MEK, a downstream kinase from RAS, in KRAS mutant non-small cell lung cancer [26]. However, the combination of gefitinib (EGFR inhibitor) and trametinib (MEK inhibitor) didn’t do any better than the single agents, both in experiments performed in lung and colorectal cancer. So, Rene moved his research forward by employing the same tactic as for solving the issue of BRAF inhibition. This time, the genetic screen revealed ERBB3, a member of the EGFR family, as the synthetic lethal event [27].

Looking into understanding why MEK inhibitors are not effective on their own, Rene’s lab found out that ERBB3 becomes active as a consequence of MEK inhibition, and will signal through RAS and MEK, rendering MEK inhibition on its own insufficient to completely inhibit the pathway [27].

Based on this, they hypothesized that pan HER inhibitors, small molecules that inhibit all members of the ERB family and not only EGFR, would improve the clinical result of MEK inhibitors. Following promising results in in vitro and in vivo pre-clinical studies using various pan HER inhibitors [27], the research was advanced to clinical trials where three different MEK inhibitors and three different Pan HER inhibitors were tested. Unfortunately, in the clinical trials, it proved impossible to balance the tradeoff between toxicity and efficacy [28-30]. Rene recalls in the webinar that they “were never able to reach an effective concentration to inhibit the pathway because we saw toxicity long before we saw efficacy in these patients.”

2.1.3. KRAS inhibitors: adapting lung cancer treatments to fight colorectal cancer

Nowadays, we have drugs that target one level higher than MEK, molecules bind and inactivate RAS directly, more precisely KRAS G12C selective inhibitors [31]. These drugs have been thought to be quite effective in lung cancer, but not in colon cancer [32]. Once again, the Bernards and Bardelli labs returned to the concept that EGFR inhibitors could potentially be highly synergistic with drugs that act further downstream in the MAP kinase pathway, and alongside other researchers such as Sandra Misale, for instance, decided to test a combination of EGFR inhibitors and KRASG12C inhibitor. This combination proved highly synergistic both in lung and colon cancer throughout experiments, from in vitro systems to clinical practice (colon cancer) [33, 34].

This raised the question of why RAS inhibition with EGFR is effective, but MEK inhibition with EGFR is ineffective in the same setting. Understanding the molecular mechanisms at play is an important focus of the Bernards Lab at the moment.

2.1.4. KRAS inhibitors: what doesn’t kill them makes them stronger

Although promising at first, KRAS G12C inhibitors have proved to be only limitedly effective in lung cancer, similar to what was previously observed following treatment with chemotherapeutic agents. Even though the initial response to treatment is stronger following RAS inhibition, tumours quickly develop resistance and undergo a boosted progression afterwards [35].

In the meantime, research in Prof Bernards’ lab uncovered another kinase with clinical potential on the MAPK pathway. Rene recalls “we more or less by coincidence, found that if you inactivate MAP2K4 in KRAS lung cancer cells, the genetic inactivation of MAP2K4 made a cell sensitive to MEK or ERK inhibitors”. Investigating the molecular mechanisms, they revealed that ERBB activation following MEK inhibition comes as a feedback activation of the MAP2K4-JNK-JUN pathway as the two pathways are connected by a dual specific phosphatase called DUSP4 [36]. Inactivation of MAP2K4 also improves the effectiveness of pan-RAS inhibitors and KRAS G12C inhibitors, such as Sotorasib [37]. This suggests that by acting on this parallel pathway that limits the response to the conventional MAP kinase inhibition, MAP2K4 inhibitors have the potential to improve the effectiveness of a broad range of drugs that act on the MAPK pathway.

Stefan Laufer and Lars Zender working on liver regeneration made a small molecule MAP2K4 inhibitor to enhance liver regeneration [38, 39]. As predicted, this inhibitor has indeed proven to be highly synergistic with MEK and KRAS inhibitors in pre-clinical studies in both lung and colorectal cancer, suggesting that their MAP2K4 inhibitor could be repurposed for cancer treatment [37].

2.2. Prof. Barbacid’s most recent adventures with the MAPK pathway for the improvement of pancreatic cancer treatment

Several RAS inhibitors, both pan-RAS and G12-specific inhibitors, have been developed to date [31, 40-43], but their effectiveness in pancreatic cancer has been limited [41, 44]. Thus, similar to lung and colorectal cancer, the treatment of pancreatic cancer is unlikely to be majorly impacted by current KRAS inhibitors used as single agents.

Prof Barbacid hypothesized that targeting MAPK signaling simultaneously at multiple levels could present a more effective alternative to targeting KRAS directly. However, while promising to potentially improve initial response and reduce the risk of developing resistance to the treatment in pancreatic cancer patients, such approaches also run the risks of generating higher and potentially intolerable toxicity.

One approach that the Barbacid lab tested was a concomitant inhibition of EGFR and C-RAF. Pre-clinical studies showed complete regression of a subset of pancreatic cancer cells and obstructed progression of patient-derived xenografts bearing the same mutation without apparent toxicity concerns [45]. Whilst systemic deletion of Egfr and Raf1 in both Kras/Trp53-driven GEM tumour models as well as in human PDXs models led to complete regression in a subset of the tumours, worryingly some of the tumours were resistant to EGFR/RAF1 ablation. Efforts to decipher the molecular mechanisms through which this combination works revealed crosstalk with the JAK-STAT3 signaling pathway: IL6-JAK-STAT3 signalling was upregulated in response to EGFR/RAF1 ablation. Digging a bit further Prof. Barbacid’s lab was able to pin down resistance to EGFR/RAF1 inhibition to the selective phosphorylation of STAT3 at tyrosine 705 [45].

Initial in vitro experiments showed that inhibition of STAT3 expression eliminates resistance to RAF1/EGFR ablation. Further experiments in GEM-derived orthotopic PDAC Models showed that concomitant ablation of these three signaling nodes: RAF1, EGFR and STAT3, resulted in an impressive complete elimination of pancreatic tumors with no signs of relapse during the duration of the experiment [46]. Current efforts in the Barbacid lab are directed towards the pharmacological validation of this novel therapeutic strategy. This comes with a series of new challenges, but preliminary results from KRAS/P53 mutated PDAC patient-derived organoid and patient-derived xenograft cultures effectively blocked tumour progression, bringing the promise of improved targeted therapies for PDAC patients [46].

2.3. Concluding remarks

What we’ve learned from Prof. Bernards’ and Prof. Barbacid’s research over the past 20 years is that when it comes to targeting the MAPK pathway, combinatorial therapies promise to be more effective in killing cancer cells than single-agent targeted therapies. However, balancing the anti-tumoural effect and increased toxicity in patients is a very challenging task. Two promising approaches at the moment are a triple STAT3/ RAF1/ EGFR inhibition and MAP2K4 inhibition that has the potential to be combined with the inhibition of various other players on the MAPK pathway.

Although our knowledge of the function of the MAPK pathway in homeostasis and cancer progression has drastically improved over the past 40 years, there are still gaps in our knowledge waiting to be filled. For instance, more research needs to be done to understand the molecular mechanisms at play behind the effectiveness of RAS/ EGFR inhibition and the lack thereof in MEK/ EGFR inhibition in lung and colon cancer. Similarly, the molecular mechanism responsible for tumour ablation in pancreatic cancer following EGFR/ RAF1/ STAT3 inhibition and the lack of response following individual inhibition of each signaling node is not fully understood at the moment and represents an avenue for future research.

Whilst this article and the lectures that inspired it focused on lung, colorectal and pancreatic cancer, attention should be directed towards other RAS-driven cancers as well.

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