Pancreatic adenocarcinoma (PDAC) is one of the deadliest cancer types, with a 5-year survival rate of only 10% [1]. Due to the lack of specific early symptoms, the majority of the patients are diagnosed at an advanced disease stage with already established metastasis. Moreover, mostly due to the lack of targetable molecular alterations, surgical removal and conventional chemotherapy are the only available treatment options for PDAC patients [1].

Although a number of driver mutations have been identified in PDAC tumors in addition to several tumor suppressors [CDKN2A (cyclin-dependent kinase inhibitor 2A), TP53 (tumor protein p53)] and the predominant KRAS (Kirsten Rat Sarcoma) mutations, the percentage of patients with therapeutically targetable mutations is low [2]. One exception is the relatively rare BRCA1/2 (Breast Cancer type 1/2 susceptibility protein) mutation which renders tumor cells sensitive to PARP [Poly (ADP-ribose) polymerase] inhibitors [3]. Furthermore, PDACs are generally stroma-rich, “cold” tumors with low immunogenicity, in which immunotherapeutic modalities provide no clinical benefit [4] with the exception of the rare occurrence of MMR deficiency which results in sensitivity to immunotherapy [5].

Approximately 90% of PDAC tumors harbor an activating mutation in the KRAS proto-oncogene [6], among which the most frequently present is the G12D mutation at ∼50%, G12V at ∼30% and G12R at 10% while other mutations do not reach the frequency of 5% [7]. In PDAC, G12D mutant KRAS is considered to be a major driver in contrast to lung or colorectal cancer [8]. Mutant KRAS has long been considered “undruggable” due to its compact structure and specific molecular properties, but the recent introduction of allele-specific inhibitors has challenged this label [9]. Covalent inhibitors of the G12C mutant KRAS have already been approved for clinical application [10]. Nevertheless, the vast majority of G12C mutations are mainly linked to lung adenocarcinoma [7]. Clinically available therapeutic options for tumors with other forms of KRAS mutations are yet to be developed. Notably, there are several established KRAS G12D inhibitors available, and even the first reports on the possibility of covalent targeting of KRAS G12D have been published [11]. Among the new KRAS G12D inhibitors, MRTX1133 is the first-in-class specific, non-covalent inhibitor of the KRAS G12D oncoprotein [12]. This small-molecule inhibitor shows high selectivity for the G12D form of KRAS over the wild-type protein, preventing effector protein binding and blocking subsequent cell signaling activation [13]. MRTX1133 can inhibit the viability of KRAS G12D mutation harboring cell lines at nanomolar concentrations and it also shows potent in vivo activity [13].

The treatment of KRAS-mutant tumors has been dramatically shifted with recent advances in targeted therapies. However, evidence of acquired or de novo resistance to covalent G12C inhibitors has already been demonstrated, with only approximately 40% of patients showing partial response to KRAS-targeting monotherapies in lung adenocarcinoma and less than 20% in colorectal cancer [14]. Several resistance mechanisms have been already described, ranging from new mutations in the KRAS gene to the rewiring of the tumor cells’ signalization network [14, 15]. The latter is most evident in the feedback reactivation of RAS signaling by wild-type RAS proteins (HRAS, NRAS, or the wild-type KRAS allele in the case of heterozygous KRAS mutations) [15]. Additionally, resistance mechanisms revealed in KRAS G12C mutant tumors have been observed following MRTX1133 treatment in G12D mutant cancer models. Upstream activation of ERBB (erythroblastic leukemia viral oncogene homolog) receptors after MRTX1133 exposure has also been shown in colorectal and pancreatic cancers, leading to sustained wild-type RAS signaling [16–18]. Upregulation of mammalian target of rapamycin (mTOR) signaling, epithelial-to-mesenchymal transitions, or specific gene amplifications have also been observed [19].

Thus, several combination approaches have been proposed, most of which focus on the concomitant targeting of mutant KRAS and reactivation of wild-type RAS signaling. These include the combination of KRAS G12C inhibitors with therapeutic modalities targeting upstream elements of the RAS pathway like EGFR (epidermal growth factor receptor) -blocking antibodies or SOS1 (son of sevenless 1) inhibitors in addition to targeting SHP2 (Src homology 2-containing protein tyrosine phosphatase 2), which is also a positive modulator of RAS signaling [20]. The application of immunomodulatory therapies, cell cycle inhibitors, or horizontal targeting of the PI3K-AKT pathway in combination with KRAS G12C inhibitors is also being investigated [20]. As the more effective, covalent targeting of G12D is much harder than in the case of the G12C mutant KRAS, and the first-in-class drug MRTX1133 is also a non-covalent drug, the application of combinational therapy seems to be rational.

Our group has previously shown that the combination of KRAS G12C inhibitors with farnesyl-transferase inhibitors exerts synergistic anti-tumoral effects in several KRAS G12C tumor models, including pancreatic tumor cells [21]. As farnesylation is a key post-translational modification of multiple important regulatory proteins, the addition of farnesyl-transferase inhibitors appears to integrate the advantages of agents used in various KRAS combination therapies. These include inhibition of feedback reactivation of HRAS, horizontal targeting of the PI3K-AKT pathway by RHEB (ras homolog enriched in brain), and cell cycle arrest by inducing defects in the nuclear lamina and CENPE/F (centromere associated protein E/F) proteins [21].

Here, we describe that this combination strategy facilitates the anti-tumoral activity of KRAS G12D inhibition and successfully breaks down therapeutic resistance in pancreatic adenocarcinoma.

In the past few years, intensive work by the research community has led to breakthroughs in the treatment of KRAS-mutant cancers, especially in the G12C mutant form [20]. KRAS was considered undruggable due to its compact form, picomolar affinity for GTP (Guanosine-5′-triphosphate), and high homology between the different RAS genes [20]. Therefore, an activating mutation in this proto-oncogene simply served as an exclusion criterion for any targeted therapy, like EGFR-targeting agents [7]. Thus, the development of allele-specific inhibitors of KRAS represents a major step forward, especially considering the positive responses to these agents observed in the clinic. Although these clinically observed anti-tumoral activities are unprecedented in treating KRAS mutant tumors, the majority of patients still show intrinsic resistance to KRAS inhibitors [14]. For this reason, combination settings are urgently needed to break down resistance and to increase the number of patients that show response. Accordingly, various combination therapies are being tested, including the combination of KRAS G12C inhibitors with EGFR, SOS1, SHP2, and cell cycle inhibitors or horizontal targeting of the AKT-mTOR pathway, the majority of which are currently being investigated in clinical trials [20].

Recently, we have shown that the combination of farnesyl-transferase inhibition with KRAS G12C inhibitors results in synergistic antitumoral effects, independent of the tissue of origin in 2D, 3D, and in vivo models [21]. We have demonstrated that the addition of farnesyl-transferase inhibitors to KRAS targeting interferes with multiple signaling mechanisms, like feedback reactivation of RAS signaling through wild-type HRAS proteins, AKT-mTOR signaling by blockade of farnesylated RHEB, or blocking cell cycle progression by interfering with the laminar network and farnesylation of CENPE/F [21]. Notably, most of these mechanisms overlap with the currently investigated combinational approaches targeting oncogenic and wild-type RAS signaling in parallel (KRAS G12C inhibitors plus EGFR, SOS1, or SHP2 targeting) or with inhibitors of the cell cycle or the AKT-mTOR pathway [15, 20].

Here, we have demonstrated that the application of tipifarnib, one of the most potent farnesyl-transferase inhibitors results in synergistic anticancer effects in combination with KRAS G12D targeting pancreatic adenocarcinoma cells that have been shown to be therapy-resistant under 2D conditions.

In our panel of pancreatic adenocarcinoma cell lines, PANC1, a well-known model of human PDAC, showed resistance to MRTX1133 single agent treatment (IC50 > 3,000 nM) in contrast with ASPC1 (IC50 = 21.9 nM) and SW1990 (IC50 = 11.6 nM). In assessing changes in RAS-related signaling after MRTX1133 treatment, we observed strong inhibition of ERK and S6 activation, key members of the two most-studied RAS-regulated pathways. The most unique - although not statistically significant - change in the therapy-resistant cell line was the elevation in KRAS expression after the MRTX1133 application. As this phenomenon could not be observed in the other cell lines, it may reflect a compensatory resistance mechanism.

Next, we sought to examine whether combining tipifarnib with MRTX1133 could overcome the resistance of the PANC1 cell line in vitro. Notably, the combination therapy in the 2D model resulted in synergistic anti-cancer effects with a Loewe score of 12. The strongest effect could be seen when the highest MRTX1133 dose was combined with a lower tipifarnib concentration. Additionally, the combination of MRTX1133 with lonafarnib, an alternative farnesyl-transferase inhibitor, also showed synergistic drug interactions, especially at higher FTi concentrations.

We also evaluated the efficacy of combination therapy in PANC1 tumor spheroids. In line with previous findings by others [24], we observed a dramatic change in sensitivity to KRAS targeting when we compared 2D to 3D conditions, more specifically, from IC50 > 3,000 nM in 2D conditions to IC50 = 4.8 nM in the 3D environment. Differential effects of 3D compared to 2D culture conditions are a well-known phenomenon in preclinical cancer research and 3D culture is considered to be more representative of the in vivo situation [25, 26]. Notably, it has been shown that culturing cells in spheroids enhances the effects of chemotherapeutic agents [27], or sensitizes towards KRAS targeting [24, 28]. We also have demonstrated in our previous work that KRAS G12C mutant models are more prone to direct KRAS targeting in 3D or in vivo compared to 2D monoculture [21]. Unfortunately, the other 2 cell lines were not able to grow into spheroids in our settings, therefore the sensitivity of PANC1 to MRTX1133 could not be directly compared to them in 3D.

Still, the addition of tipifarnib resulted in synergism with a Loewe score of 11.5, although the most synergistic area in the synergy map visualizing synergy scores has shifted to higher tipifarnib concentrations combined with higher MRTX1133 doses.

Examination of RAS signaling revealed that tipifarnib and combination therapy elevated the level of activated AKT while reducing the phosphorylation of S6, which is a downstream target of AKT. This phenomenon can be partly explained by inhibition of RHEB farnesylation as well as crosstalk between the RAS/MEK/ERK pathway and the PI3K-AKT-mTOR signaling [29]. Indeed, we demonstrated that tipifarnib and combination therapy successfully blocked RHEB farnesylation, leading to the accumulation of its non-farnesylated, non-functional form. ERK activation was also significantly increased after tipifarnib treatment; however, combination therapy reduced ERK phosphorylation similar to MRTX1133 monotherapy.

Investigation of HRAS revealed that tipifarnib and combination therapy depleted functional, farnesylated HRAS by interfering with its farnesylation. Previously, we have shown that KRAS targeting results in compensatory reactivation of wild-type RAS proteins, more specifically, overactivation of HRAS induced by KRAS G12C inhibitors [21]. Thus, successful inhibition of its farnesylation can also abrogate this resistance mechanism in KRAS G12D targeting.

We also assessed changes in KRAS expression, although this protein is not sensitive to farnesylation inhibition due to alternative geranyl-geranylation [30]. In line with this, no upward shift could be observed after farnesyl-transferase inhibitor treatment. Interestingly, although not statistically significant, visible trends in changes in KRAS expression could be observed, with the combination treatment reducing KRAS levels not only compared to MRTX1133 monotherapy, which increased them, but also compared to the control. Although the mechanism behind this phenomenon is not known, it may contribute to the observed synergy between the two drugs.

No apoptosis could be detected in any of the treatments based on the level of cleaved PARP. This could be explained by the fact that PANC1 has homozygous mutations in TP53 (p.Arg273His), a key regulator of apoptosis. Interestingly, the expression of p-Histone H3, a known marker of M-phase cells was decreased by MRTX1133 and combination therapy (the latter change was not statistically significant), whereas tipifarnib monotherapy slightly increased its expression. This finding is in line with our previous observation, where we showed that farnesyl-transferase inhibition leads to defects in cell division, leading to the accumulation of M-phase cells [21]. Interestingly, when we analyzed the cell cycle distribution after a longer, 96-hour treatment, we observed a decrease in the S-phase cell ratio after MRTX1133 and combination therapy, with a more pronounced effect of the latter. In line with our previous findings, we found that tipifarnib and combination therapy led to an accumulation of cells with aberrant, segmented nuclear morphology, indicating defects in the mitotic process [21].

In summary, we have demonstrated that MRTX1133 sensitivity shows high variability in KRAS G12D mutant pancreatic adenocarcinoma models in 2D culture. Further investigation of the resistant cell model revealed that the addition of a farnesyl-transferase inhibitor to MRTX1133 can abrogate this resistance, leading to synergistic antitumoral effects. Interestingly, culturing cells in spheroid culture dramatically sensitized resistant cells to MRTX1133 treatment. Nevertheless the synergistic effects of combination treatment are also maintained in the 3D environment.

Regarding the mechanisms behind the synergistic effects, we identified elevated KRAS expression after MRTX1133 treatment, which was ultimately decreased by combination therapy. Additionally, similar to our previous observations, we found that inhibition of farnesylation of RHEB and HRAS may contribute to synergistic effects.

Our findings warrant further investigation of the applicability of KRAS G12D targeting in combination with farnesyl-transferase inhibitors in KRAS G12D mutant pancreatic adenocarcinoma.