# Ras Isoforms from Lab Benches to Lives--What Are We Missing and How Far Are We?
## 6 Future Recommendations for Devising Isoform-Specific Targeting
Early clinical trials as reported by Amgen show the partial response of diminished tumor size in patients with non-small-cell lung cancer (NSCLC). Mirati Therapeutics also reported similar effects on the administration of MRTX489 to NSCLC, but the results in CRC patients in both cases were less promising. Although K-Ras signaling was attenuated in cell lines treated with MRTX849 and AMG510, the degree of sensitivity to these inhibitors varied between cell lines. A combinatorial therapy using AMG510 and a MEK inhibitor showedaugmented efficacy of treatment. A major limitation of these two inhibitors is that neither AMG510 nor MRTX849 could affect PI3K signaling. PI3K is one important effector of Ras isoforms and it activates Akt family Ser/Thr kinases crucial for inhibition of apoptosis and cell survival [169]. Its importance in K-Ras signaling cannot be overlooked. To fully realize the potential of such K-Ras inhibitors additional combination strategies, as well as efficient targeting of most if not all signaling pathways downstream to K-Ras are to be addressed. As mentioned in the above sections compensatory or alternative mechanisms exist to compensate for the loss of a functional protein. This leads to acquired resistance to inhibitors. For instance- resistance against anaplastic lymphoma kinase (ALK) and EGFR inhibitors developed within 1-2 years of treatment because of mutations at drug binding sites and activation of alternative RTKs that continued downstream signaling [170]. Other K-Ras covalent inhibitors currently in a clinical trial include JNI-74699157 (ARS-3248), LY3499446, etc. [171]. [PERSON] et al., obtained KRpep-2 (Ac-RRCPLYISYDPVCRR-NH2) through random screening of phage-displayed peptide libraries against K-Ras G12D. KR pep-2 selectively inhibited K-Ras G12D and significantly suppressed phosphorylation of ERK. A 30 mM concentration, KRpep-2 inhibited the proliferation of cancer cell line A427 as well [172]. Another approach used was to develop two recombinant peptides using amino acids 5-21 of K-Ras G12D (SP2) coupled to _Diptheria_ toxin (DTT) as a carrier. Both these peptides DTT-SP4 and DTSP when administered as vaccines in combination with alum and cytosine phosphoguanine (CpG) gave rise to SP-specific humoral and cell-mediated immune response in CT26 mouse tumor models [173]. [PERSON] et al. identified four CD8+ T cell clonotypes specific for K-Ras G12D from infiltrating lymphocyte population in metastasizing colorectal cancer. Adoptive transfer of these infiltrating lymphocytes leads to regression of lung metastasis in all 7 lung lesions. However, one lesion out of these 7 (lesion3) showed progression due to the loss of chromosome 6 haplotype that encodes for class I MHC molecule required for tumor recognition by K-Ras G12D-specific T cells [174]. Hence the adoptive transfer of tumor-infiltrating lymphocytes (TIL) offers a promising therapeutic approach against codon-specific mutation. The limitation of this approach lies in the fact that TIL only recognizes patient-specific antigens, and thus requires patient-specific T cells for each therapy. Moderna (Cambridge, MA, USA) and Merck (Darmstadt, Germany) are currently collaborating to test mRNAs vaccines against K-Ras tumors. mRNA act as a delivery vector to introduce tumor-specific antigens (TSA) and tumor-associated antigens (TAA) into cells. Once expressed and presented by MHCs, these antigens can mount both B Cell-mediated humoral as well as and CD4+/CD8+ cytotoxic immunity to clear malignant cells. A combinatorial therapy for pancreatic cancer using mRNA-5671 (encoding K-Ras neoantigen) with KEYTRUDA (a PD-1 specific inhibitor) is currently under clinical trial [175]. Although these studies open up the possibility of targeting G12D mutants, it yet remains in only a preliminary state of therapeutic plausibility. K-Ras G12V mutants with a very low intrinsic GTPase activity account for about 30% of all Ras mutations in tumors. It remains active for a very long period [176].