Background: Oncogenic alteration in RET, representing 1-2% of NSCLC, is an important target. Pralsetinib is a selective RET inhibitor targeting RET fusions. We present a case series on opportunistic infections and the mechanism of immunosuppression. Methods: From October 2021 to March 2022, we administered pralsetinib to a total of 15 patients with NSCLC harboring RET fusion. We retrospectively analyzed the clinical efficacy and adverse events related to pralsetinib. To investigate pralsetinib’s impact on T-cells, we examined cytokine release using ELISA from Jurkat T (JT) cells after pralsetinib treatment. To test if Stat5 binds to the IL-2 promoter, we conducted Chromatin Immunoprecipitation (ChIP). Results: Out of 18 patients with measurable disease, 14 (93%) achieved a partial response. With a median follow-up duration of 8.7 months, four cases of opportunistic infections occurred. Notably, three patients (16.6%) experienced invasive pulmonary aspergillosis, and one patient (5.5%) experienced cytomegalovirus pneumonia. To investigate mechanisms behind these observations, we aimed to replicate conditions similar to those of activated T cells. Thus, JT cells were treated with PMA and ionomycin to induce IL-2 release through NFAT, AP-1, and NF-κB binding to the IL-2 promoter. After ∼2 weeks of pralsetinib treatment, Jak3 signaling was inhibited, leading to decreased IL-2 release by downregulating transcription factors associated with IL-2 production, such as JunB/c-Jun and Stat5, which are downstream components of Jak3 signaling. Additionally, to investigate the direct correlation between Jak3 and IL-2 release, JT cells were treated with ritlecitinib, a Jak3-selective inhibitor, for ∼2 weeks. Like pralsetinib, a reduction in IL-2 release was observed. Furthermore, based on the evidence that Stat5 inhibition leads to reduced IL-2 release, we showed that Stat5, a direct downstream component of Jak3, binds to the IL-2 promoter during activation of Jurkat T cells. Conclusion: Pralsetinib, not Selpercatinib, inhibits additional IL-2 production by blocking Jak3/Stat5 activation triggered by IL-2 released during early T cell activation. Consequently, Jak3 inhibition by pralsetinib suppresses IL-2 release by inhibiting the activation of transcription factors for IL-2, such as JunB/c-Jun and Stat5, thereby inducing opportunistic infections, including invasive pulmonary aspergillosis (IPA), cytomegalovirus (CMV) pneumonia, CMV viremia, and pneumocystis pneumonia. Citation Format: Shinkyo Yoon, Hyun-Min Ryu, Eunjin Lee, Yunkyung Sung, Ho-Su Lee, Ji Eun Park, Chang Hoon Lee, Seyoung Seo, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. Pralsetinib induces opportunistic infection in RET fusion-positive NSCLC patients via inhibition of IL-2 production by blocking Jak3/Stat5 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3112.

Background: Oncogenic alteration in RET, representing 1-2% of NSCLC, is an important target. Pralsetinib is a selective RET inhibitor targeting RET fusions. We present a case series on opportunistic infections and the mechanism of immunosuppression. Methods: From October 2021 to March 2022, we administered pralsetinib to a total of 15 patients with NSCLC harboring RET fusion. We retrospectively analyzed the clinical efficacy and adverse events related to pralsetinib. To investigate pralsetinib’s impact on T-cells, we examined cytokine release using ELISA from Jurkat T (JT) cells after pralsetinib treatment. To test if Stat5 binds to the IL-2 promoter, we conducted Chromatin Immunoprecipitation (ChIP). Results: Out of 18 patients with measurable disease, 14 (93%) achieved a partial response. With a median follow-up duration of 8.7 months, four cases of opportunistic infections occurred. Notably, three patients (16.6%) experienced invasive pulmonary aspergillosis, and one patient (5.5%) experienced cytomegalovirus pneumonia. To investigate mechanisms behind these observations, we aimed to replicate conditions similar to those of activated T cells. Thus, JT cells were treated with PMA and ionomycin to induce IL-2 release through NFAT, AP-1, and NF-κB binding to the IL-2 promoter. After ∼2 weeks of pralsetinib treatment, Jak3 signaling was inhibited, leading to decreased IL-2 release by downregulating transcription factors associated with IL-2 production, such as JunB/c-Jun and Stat5, which are downstream components of Jak3 signaling. Additionally, to investigate the direct correlation between Jak3 and IL-2 release, JT cells were treated with ritlecitinib, a Jak3-selective inhibitor, for ∼2 weeks. Like pralsetinib, a reduction in IL-2 release was observed. Furthermore, based on the evidence that Stat5 inhibition leads to reduced IL-2 release, we showed that Stat5, a direct downstream component of Jak3, binds to the IL-2 promoter during activation of Jurkat T cells. Conclusion: Pralsetinib, not Selpercatinib, inhibits additional IL-2 production by blocking Jak3/Stat5 activation triggered by IL-2 released during early T cell activation. Consequently, Jak3 inhibition by pralsetinib suppresses IL-2 release by inhibiting the activation of transcription factors for IL-2, such as JunB/c-Jun and Stat5, thereby inducing opportunistic infections, including invasive pulmonary aspergillosis (IPA), cytomegalovirus (CMV) pneumonia, CMV viremia, and pneumocystis pneumonia. Citation Format: Shinkyo Yoon, Hyun-Min Ryu, Eunjin Lee, Yunkyung Sung, Ho-Su Lee, Ji Eun Park, Chang Hoon Lee, Seyoung Seo, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. Pralsetinib induces opportunistic infection in RET fusion-positive NSCLC patients via inhibition of IL-2 production by blocking Jak3/Stat5 activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3112.

Hepatocellular carcinoma (HCC) comprises the majority of primary liver cancers and possesses a low 5-year survival rate when in the advanced stages. Anillin (ANLN), a key player in cell growth and cytokinesis, is implicated in HCC development. Currently, no treatment agents are known to suppress ANLN. Analysis of The Cancer Genome Atlas data showed that high ANLN expression is associated with poor prognosis and survival in HCC patients. ANLN knockdown was shown to inhibit proliferation, cell cycle progression, and PD-L1 expression in liver cancer cells. The antipsychotic drug penfluridol was identified to suppress ANLN expression in the Connectivity Map analysis. Penfluridol downregulated ANLN at both the mRNA and protein levels, leading to G2/M cell cycle arrest and reduced colony formation in liver cancer cells. Mechanistically, penfluridol inhibited the transcription factor MYC from binding to an E-box motif in the ANLN promoter. This process was mediated by penfluridol-induced upregulation of NRF2, which competitively bound and sequestered MYC away from the ANLN promoter. Penfluridol inhibited the interaction between NRF2 and KEAP1, increasing NRF2. In a syngeneic mouse model, penfluridol suppressed liver tumour growth accompanied by increased NRF2 and decreased MYC and ANLN expression. These findings suggest penfluridol can be applied as the first ANLN blocker to modulate the MYC/NRF2/KEAP1 axis.

Abstract Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of non-small cell lung cancer (NSCLC), but the predictive value of PD-L1 expression alone remains limited. Thus, there is a need for additional predictive biomarkers and functional assays to guide ICI use more precisely. Recent clinical observations suggest that NSCLC patients harboring MED12 mutations tend to respond more favorably to ICI treatment. Based on prior studies reporting that MED12 mutations can lead to functional loss of the MED12 protein, we generated MED12 knock-out NSCLC cell lines using CRISPR-Cas9 and observed significantly increased PD-L1 expression compared to control cells, as confirmed by western blotting and flow cytometry. To evaluate functional sensitivity to ICI, we developed a novel in vitro co-culture system using CAR-engineered Jurkat T cells. In this system, MED12-deficient NSCLC cells suppressed Jurkat T cell activation, which was restored upon treatment with atezolizumab, an anti–PD-L1 antibody. These results demonstrate that PD-L1 upregulation caused by MED12 loss leads to T cell inhibition, which can be reversed with PD-L1 blockade. In addition, we found that the regulation of PD-L1 expression is associated with altered YAP activity following the loss of MED12 function. This finding suggests that the YAP signaling pathway may serve as a key mediator of immune microenvironment changes in MED12-mutated tumors. In conclusion, our study is the first to demonstrate that MED12 mutation promotes an immunosuppressive environment through PD-L1 upregulation and alters ICI responsiveness in NSCLC. MED12 thus emerges as a promising predictive biomarker for ICI treatment outcomes. Furthermore, our CAR–Jurkat T cell–based in vitro assay offers a practical and quantitative platform for preclinical screening of ICI efficacy, with potential for broad application in immuno-oncology research and drug development. Citation Format: Hyun-Min Ryu, Deokhoon Kim, Shinkyo Yoon, Yunkyung Sung, Ho-Su Lee, Ji Eun Park, Chang Hoon Lee, Wanlim Kim, Seyoung Seo, Sang-We Kim, Kang-Seo Park, Dae Ho Lee. MED12 loss promotes PD-L1–mediated immune evasion and predicts response to immune checkpoint inhibitors in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C062.

Non-small cell lung cancer (NSCLC) is frequently associated with mutations in receptor tyrosine kinases (RTKs), such as EGFR and ALK. While RTK inhibitors (RTKIs) have proven effective in treating patients with specific RTK mutations, the emergence of resistance to these therapies remains a significant clinical obstacle. As such, there is still an unmet need for the identification of new biomarkers that can predict resistance to RTK inhibitors in clinical use. In the present study, we demonstrate that MED12 mutations are a key driver of RTKi resistance in NSCLC cells. This resistance is mediated through the release of inflammatory cytokines triggered by MED12 degradation. Notably, we observed that of the two major downstream signaling pathways activated by inflammatory cytokines, only the MEK/ERK pathway was upregulated, while the PI3K/AKT pathway was unaffected in MED12 knock-out (KO) cells. The degradation of MED12 results in the dissociation of the MED12 complex, which subsequently leads to YAP phosphorylation. This phosphorylated YAP increases PTEN expression by inhibiting miR-29, thereby suppressing the PI3K/AKT signaling pathway. Importantly, treatment with trametinib, a MEK inhibitor, effectively suppressed tumor growth in MED12KO NSCLC cells and in xenograft models derived from these cells. These findings suggest that targeting the MEK/ERK signaling pathway, such as with trametinib, may provide a viable strategy to overcome RTKi resistance in MED12-mutant NSCLC. Furthermore, MED12 is identified as a crucial biomarker and potential therapeutic target for overcoming RTKi resistance.

SPC induces ZEB1 phosphorylation, with ERK2, rather than ERK1, targeting the S322 site. Inhibiting S322 phosphorylation mitigates EMT, PD-L1 expression, and progression of pancreatic cancer. The phosphoS322 detection antibody might be a valuable tool for predicting pancreatic cancer prognosis. These findings implicate ERK2 as a potential therapeutic target for pancreatic cancer and highlight phosphoZEB1 as a valuable prognostic marker.

Histone H3 lysine 36 (H3K36) methylation, a pivotal epigenetic mark that ensures transcriptional fidelity and genomic integrity, plays an essential role in development and tumorigenesis. The nuclear receptor-binding SET domain (NSD) family of histone methyltransferases, comprising NSD1, NSD2, and NSD3, primarily catalyzes mono- and di-methylation of H3K36 (H3K36me1/2) and engages with chromatin-associated and transcriptional regulatory complexes in a context-dependent manner. Increasing evidence demonstrates that NSD family members have emerged as critical drivers in human cancers. Recurrent gene amplifications, point mutations, and oncogenic fusions of NSD family genes are frequently observed in both solid and hematologic cancers. Their dysregulation contributes to tumorigenesis, cancer cell proliferation and survival, and metastatic progression through both H3K36 methylation-dependent and -independent mechanisms. Pharmacological inhibition of NSD catalytic activity, as well as alternative approaches such as targeted protein degradation or disruption of cofactor interactions, are emerging as promising therapeutic strategies for cancer treatment. This review summarizes the structural features, molecular functions, and cancer-associated alterations and mechanisms of the NSD family and highlights recent advances in targeting these enzymes as potential epigenetic vulnerabilities in cancer.