Progress and challenges in RET-targeted cancer therapy.

The rearranged during transfection (RET) is a receptor protein tyrosine kinase. Oncogenic RET fusions or mutations are found most often in non-small cell lung cancer (NSCLC) and in thyroid cancer, but also increasingly in various types of cancers at low rates. In the last few years, two potent and selective RET protein tyrosine kinase inhibitors (TKIs), pralsetinib (BLU-667) and selpercatinib (LOXO-292, LY3527723) were developed and received regulatory approval. Although pralsetinib and selpercatinib gave high overall response rates (ORRs), < 10% of patients achieved a complete response (CR). The RET TKI-tolerated residual tumors inevitably develop resistance by secondary target mutations, acquired alternative oncogenes, or MET amplification. RET G810 mutations located at the kinase solvent front site were identified as the major on-target mechanism of acquired resistance to both selpercatinib and pralsetinib. Several next-generation of RET TKIs capable of inhibiting the selpercatinib/pralsetinib-resistant RET mutants have progressed to clinical trials. However, it is likely that new TKI-adapted RET mutations will emerge to cause resistance to these next-generation of RET TKIs. Solving the problem requires a better understanding of the multiple mechanisms that support the RET TKI-tolerated persisters to identify a converging point of vulnerability to devise an effective co-treatment to eliminate the residual tumors.

Targeting RET Solvent-Front Mutants with Alkynyl Nicotinamide-Based Inhibitors.

Selpercatinib (LOXO292) and pralsetinib (BLU667) are RET protein tyrosine kinase inhibitors (TKIs) recently approved for treating RET-altered cancers. However, RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET TKIs. While acquired RET G810C/R/S/V mutations were reported in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. Here, we profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution and developed novel alkynyl nicotinamide-based RET TKIs to inhibit selpercatinib/pralsetinib-resistant RET G810 mutants. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. Alkynyl nicotinamide compounds such as HSN608, HSL476, and HSL468 have better drug-like properties than alkynyl benzamides. Six of these compounds inhibited all six G810 solvent-front mutants and the V804M gatekeeper mutant with IC50 < 50 nmol/L in cell culture. Oral administration of HSN608 at a well-tolerated dose (30 mg/kg) gave plasma level > 30x the IC50s of inhibiting all G810 mutants in cell culture. In cell-derived xenograft tumors driven by KIF5B-RET (G810C) that contains the most frequently observed solvent-front mutant in selpercatinib-treated patients, HSN608, HSL476, and HSL468 significantly suppressed and caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib and identifies novel alkylnyl nicotinamide-based RET TKIs for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.

The heterogeneous transition state of resistance to RET kinase inhibitors converges on ERK1/2-driven Aurora A/B kinases.

AIM: While most patients with RET-altered cancer responded to the RET protein tyrosine kinase inhibitors (TKIs) pralsetinib (BLU667) and selpercatinib (LOXO292), few achieved a complete response. Heterogeneity in residual tumors makes it difficult to target their diverse genetic alterations individually. The aim of this study is to characterize the cancer cells that persist under continuous RET TKI treatment and identify the shared vulnerability of these cells. METHODS: We analyzed residual RET-altered cancer cells under prolonged RET TKI treatment by whole exome sequencing (WES), RNA-seq analysis, and drug-sensitivity screening. These were followed by tumor xenograft experiments of mono- and combinational drug treatments. RESULTS: BLU667- and LOXO292-tolerated persisters were cellularly heterogeneous, contained slowly proliferating cells, regained low levels of active ERK1/2, and displayed plasticity in growth rate, which we designated as in the transition state of resistance (TSR). TSR cells were genetically heterogeneous. Aurora A/B kinases were among the most significantly upregulated genes and that the MAPK pathway activity had significantly higher transcript footprints. MEK1/2 and Aurora kinase inhibitors were the most effective drugs when combined with a RET kinase inhibitor. In a TSR tumor model, combination of BLU667 with an Aurora kinase or a MEK1/2 kinase inhibitor caused TSR tumor regression. CONCLUSION: Our experiments reveal that the heterogeneous TSR cancer cells under continuous RET TKI treatment converge on the targetable ERK1/2-driven Aurora A/B kinases. The discovery of the targetable convergent point in the genetically heterogeneous TSR points to an effective combination therapy approach to eliminate the residual tumors.

The origin of exosomal miR-1246 in human cancer cells.

miR-1246 is considered an oncomiR in various cancer types. However, the origin and biogenesis of miR-1246 remain controversial which often leads to misinterpretation of its detection and biological function, and inevitably masking its mechanisms of action. Using next generation small RNA sequencing, CRISPR-Cas9 knockout, siRNA knockdown and the poly-A tailing SYBR qRT-PCR, we examined the biogenesis of exosomal miR-1246 in human cancer cell model systems. We found that miR-1246 is highly enriched in exosomes derived from human cancer cells and that it originates from RNU2-1, a small nuclear RNA and essential component of the U2 complex of the spliceosome. Knockdown of Drosha and Dicer did not reduce exosomal miR-1246 levels, indicating that exosomal miR-1246 is generated in a Drosha- and Dicer-independent manner. Direct digestion of cellular lysate by RNase A and knockdown of the RNU2-1 binding protein SmB/B' demonstrated that exosomal miR-1246 is a RNU2-1 degradation product. Furthermore, the GCAG motif present in the RUN2-1 transcript was shown to mediate miR-1246 enrichment in cancer exosomes. We conclude that exosome miR-1246 is derived from RNU2-1 degradation through a non-canonical microRNA biogenesis process. These findings reveal the origin of an oncomiR in human cancer cells, providing guidance in understanding miR-1246 detection and biological function. Abbreviations: CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; miRNA, microRNA; PDAC, pancreatic ductal adenocarcinoma; RNU2-1, U2 small nuclear RNA; RT-PCR, Reverse transcription polymerase chain reaction; sgRNA, single-guide RNA.