Microsatellite instability (MSI), tumor mutation burden (TMB), and programmed cell death ligand-1 (PD-L1) are particularly known as immunotherapy predictive biomarkers. MSI and TMB are closely related to DNA mismatch repair (MMR) pathway functionality, while the PD-L1 checkpoint mediates cancer cell evasion from immune surveillance via the PD-L1/PD-1 axis. Among all the novel triazolo[3,4-b]thiadiazole derivatives, the compound KA39 emerged as the most potent anticancer agent. In the present study, potential alterations in MSI, TMB, and/or PD-L1 expression upon cell treatment with KA39 are explored. We tested three MMR-deficient (DLD-1, LS174T, and DU-145) and two MMR-proficient (HT-29 and PC-3) human cancer cell lines. Our findings support KA39-induced PD-L1 overexpression in all cancer cell lines, although the most outstanding increase was observed in MMR-proficient HT-29 cells. MSI analysis showed that KA39 affects the MMR system, impairing its recognition or repair activity, particularly in MMR-deficient DLD-1 and DU-145 cells, enhancing oligonucleotide production. There were no remarkable alterations in the TMB between untreated and treated cells, indicating that KA39 does not belong to mutagenic agents. Taking together the significant in vitro anticancer activity with PD-L1 upregulation and MSI increase, KA39 should be investigated further for its implication in chemo-immunotherapy of cancer.