RET Inhibitor Research Articles

Abstract 5134: Immunohistochemistry expression of membrane targets for novel therapeutic agents in RET-rearranged NSCLC

Abstract Background: RET fusions are found in 1-2% of patients (pts) with advanced non-small cell lung cancer (aNSCLC). RET inhibitors (RETi) are effective, but resistance occurs. Scarce data are available on treatment after progression (PD) to RETi. Antibody-drug conjugates and bispecific antibodies are promising therapies in NSCLC, possibly employed at resistance. Data on the expression of membrane targets and the impact of novel agents are lacking. Methods: This is a multicentric retrospective study of 6 European centers. We collected archival tissue samples from pts with RET+ aNSCLC, treated or not with RETi. Membrane expression of cMET/TROP2/HER2/HER3/EGFR was centrally evaluated by immunohistochemistry (IHC). Expression was scored from 0 to 3+. cMET was assessed with EP1454Y clone, TROP2 with EPR20043, HER2 with 4B5, HER3 with D22C5, EGFR with DAKO EGFR pharmDx kit. IHC 2+ and 3+ were considered as high expression. cMET FISH testing was performed on cMET 3+ cases with available tissue. PFS was calculated from RETi start to PD or death, OS from diagnosis to death/last follow up in pts treated with RETi. Results: 52 pts were included. Median age was 65 years, 63% were female, 92% had adenocarcinoma, median PD-L1 expression was 10% (IQR 0-62.5). All samples were collected before RETi start. IHC 2/3+ scores were observed in 80% of evaluable cases (25/31) with TROP2, 60% (30/50) with cMET, 39% with HER3 (19/48), 31% with EGFR (15/48), and 4% (2/48) with HER2. 31 patients had more than 1 highly expressed target, mostly TROP2/cMET (n=17) and cMET/HER3 (n=13). FISH testing was negative for MET amplifications (N=5). PFS and OS of pts with low vs high target expression are presented in the Table. Conclusion: cMET and TROP2 were frequently expressed in RET+ aNSCLC, EGFR and HER3 were present in a lower proportion, HER2 was rarely found. These markers were not associated with a prognostic or predictive impact on RETi outcomes. The activity of novel therapeutic agents should be evaluated in this setting. PFS and OS of pts with low versus high target expression. Marker cMET TROP2 HER2 HER3 EGFR Treated with RETi, n Total=38 low=17 high=21 Total=22 low=4 high=18 Total=36 low=34 high=2 Total=37 low=24 high=13 Total=36 low=27 high=9 PFS of low vs high, median in months 12.88 vs 15.05 (HR 0.90, 95%CI 0.34-2.05, p=0.77) 3.99 vs 6.06 (HR 2.44, 95%CI 0.54-11.3, p=0.10) 13.96 vs 10.10 (HR 0.57, 95%CI 0.91-3.63, p=0.445) 7.55 vs 17.12 (HR 1.54, 95%CI 0.65-3.67, p=0.356) 12.88 vs 13.96 (HR 1.29, 95% CI 0.47-3.52, p=0.64) OS of low vs high, median in months 38.75 vs 51.6 (HR 1.29, 95%CI 0.53-3.14, p=0.55) 22.17 vs 51.6 (HR 2.09, 95%CI 0.50-8.71, p=0.19) NE 51.5 vs 62.6 (HR 1.31, 95%CI 0.50-3.46, p=0.59) 51.6 vs 62.45 (HR 0.97, 95% CI 0.32-2.96, p=0.96) Citation Format: Arianna Marinello, Maria Rosa Ghigna, Wael Salem Zrafi, Martina Mandarano, Isabelle Monnet, Carlo Genova, Alessandro Russo, Safae Terrisse, Giulio Metro, Jordi Remon, Anas Gazzah, Fabrice Barlesi, David Planchard, Jean-Yves Scoazec, Benjamin Besse, Mihaela Aldea. Immunohistochemistry expression of membrane targets for novel therapeutic agents in RET-rearranged NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5134.

Abstract 5833: Genomic mechanisms of RET inhibitor resistance in RET-fusion positive NSCLC

Abstract RET fusions are a driver oncogene detected in 1-2% of non-small cell lung cancer (NSCLC) cases. Currently, two selective RET inhibitors (RETi), selpercatinib and pralsetinib have received FDA approval for RET fusion-positive NSCLC. However, RET fusions are highly heterogeneous and the impact of specific fusion partners and breakpoints on RETi sensitivity is not well understood. Moreover, most patients eventually develop resistance to RETi, and secondary RET mutations are thought to be a key mechanism of acquired resistance. Thus, we aimed to identify RET-dependent mechanism of RETi resistance in effort to improve treatment options and clinical outcomes. We engineered a panel of Ba/F3 cells expressing various RET fusion partners and breakpoints observed in clinical cases of NSCLC and evaluated their drug sensitivity profile against a panel of RETi including selpercatinib, pralsetinib, TPX-0046, lovatinib, zeteletinib and ponatinib. We determined that individual RET fusions impart distinct sensitivities to RETi. For example, cells expressing KIF-RET (K22, R12) were sensitive to selpercatinib and pralsetinib but less sensitive to ponatinib, whereas cells expressing KIF-RET (K15, R12) were highly sensitive to selpercatinib, pralsetinib, and ponatinib. Moreover, using RET-fusion positive NSCLCs, we observed that the RETi sensitivity profile of human cell lines was similar to that observed using Ba/F3 models. To identify potential resistance mutations, we utilized the LentiMutate approach. In KIF5B-RET (K15, R12) expressing cells, we identified G810S as being associated with resistance to both selpercatinib and pralsetinib, a finding consistent with previous reports and clinical observations. In addition, we identified Y806H/N, V804M/E, and M918T as being associtated with selpercatinib-resistance and L730I/V, E732K, and Y806H/N as being associated with pralsetinib-resistance. We next constructed Ba/F3 cells expressing KIF5B-RET in combination with each secondary mutation and evaluated RETi sensitivity. G810S and Y806N conferred resistance to selpercatinib and pralsetinib, as well as to RET-targeting multi-kinase inhibitors. V804M/E mutations conferred resistance to selpercatinib but not pralsetinib, whereas L730I/V and E732K mutations conferred resistance to pralsetinib but not selpercatinib, demonstrating RETi-specific resistance mutations. Cells expressing G810S secondary mutations were resistant to selpercatinib and pralsetinib, but sensitive to zeteletinib. Collectively, we show that individual RET fusion variants have distinct drug sensitivity profiles and that secondary resistance mutations are non-overlapping between RETi. The comprehensive characterization of RET-dependent mechanisms of resistance to RETi may provide therapeutic guidance for treating RET-fusion driven NSCLC and provide structural insights that can guide the development of new therapeutic regimens. Citation Format: Ximeng Liu, Monique Nilsson, Tomohiro Takehara, Junqin He, Xiaofang Huo, Ashwani Kumar, John Heymach, Ralf Kittler. Genomic mechanisms of RET inhibitor resistance in RET-fusion positive NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5833.

Abstract 1099: Combined MEK and RET inhibition overcomes resistance to single agent RET inhibition in medullary thyroid cancer

Abstract Medullary thyroid carcinoma (MTC) is a rare neuroendocrine cancer, caused by activating mutations in the RET proto-oncogene, contributing to 13% of all thyroid cancer mortalities. Patients with metastatic MTC treated with RET inhibitors (RETi) can develop resistance through mechanisms currently being investigated. We aim to understand the molecular mechanisms of acquired resistance to RETi and develop novel combination therapies to circumvent resistance to these drugs. We used TT, an MTC cell line, to develop vandetanib-resistant (VanR) lines, at two independent sites, the Medical College of Wisconsin (MCW) and the Pediatric Oncology Branch (POB) at the NCI. We applied a multiomic approach to investigate the mechanisms of resistance, including Spectral karyotyping (SKY) and multiplex interphase Fluorescence in situ Hybridization (miFISH) to study the karyotype, ploidy, and heterogeneity at a single-cell level. Whole exome and RNA-sequencing was done to identify genetic alterations such as acquired mutations, copy number variations, and gene expression changes. In parallel, we performed genome-wide CRISPR gene knockout (KO) in TT cells treated with selpercatinib or vehicle to develop potential synergistic targeted combination therapy with RETi. Additionally, we performed a cytotoxicity screening using the Mechanism Interrogation Plate (MIPE6.0) library containing 2803 investigational drugs, novel agents, and FDA-approved drugs with known mechanisms of action. SKY and miFISH results showed that, for the parental TT_POB cell line, there were 4 copies of chromosome 10 (location of the RET gene), whereas there were 3 copies in TT_POB VanR. This was associated with an increase in the RET variant allele frequency, indicating a loss of heterozygosity of the wildtype allele as a mechanism of resistance, which was confirmed by whole exome sequencing. For TT_MCW VanR, we identified a new RET solvent front (G810S), as well as an increased copy number of RET with an increase in expression. CRISPR KO demonstrated that the knockout of NF1 led to resistance and survival to selpercatinib treatment. The MIPE6.0 screening confirmed resistance to RETi in TT_MCW VanR and sensitivity to multiple MEK inhibitors. In vivo testing in a xenograft mouse model confirmed the synergistic/additive activity of combined selpercatinib with trametinib, a MEK inhibitor. In conclusion, using a multiomic approach, we show that resistance to RETi occurs by an adaptive increase in RET signaling through secondary mutations in RET, and an increase in expression or dose of the mutant RET allele. These genetic events are associated with an activation of RAS signaling, in VanR cell lines, through the ERK/MAPK pathway. Furthermore, we demonstrate that the combination of RET and ERK/MAPK inhibition is synergistic against RET-mutant MTC. Future plans include translating our findings through clinical trials at the NCI. Citation Format: Arwa Fallatah, Abdelrahman Rahmy, Sophia Khan, Hsien-chao Chou, Kerstin Heselmeyer-Haddad, Darawalee Wangsa, David Milewski, Yong Kim, Robert Hawley, Young Song, Xiaohu Zhang, Craig Thomas, Jun S. Wei, Jong-In Park, John Glod, Javed Khan. Combined MEK and RET inhibition overcomes resistance to single agent RET inhibition in medullary thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1099.

Abstract 2857: Perspectives from a systematic review of kinase fusion oncogenes and access to corresponding kinase inhibitor clinical trials in pediatric, adolescent, and young adult solid malignancies

Abstract Oncogenic kinase fusions (KFs) have been reported in pediatric, adolescent, and young adult (P-AYA) brain tumors, sarcomas, and rare solid malignancies. Molecularly targeted therapy (MTT) matched to KFs generally produce objective responses even in advanced P-AYA malignancies. KFs occur across a broad age range of P-AYA cancer diagnoses (dx) so data on KF prevalence and the relationship between KFs and clinical characteristics including response and resistance to MTT is distributed across many studies. To address resulting knowledge gaps, we conducted a systematic review of KFs reported in P-AYA cancers and assessed the availability of clinical trials (CTs) for P-AYA patients with KF malignancies. A systematic literature search was conducted with PubMed using publication date 2013-2022, search terms for dx, age, and gene names of interest (BRAF, RAF1, NTRK1-3, ALK, FGFR1-4, MET, RET, ROS1). Known P-AYA cancer sequencing studies were also reviewed. Papers were included if they reported ≥1 patient <18 yo with a KF cancer of any type or ≤30 yo with a KF P-AYA cancer dx. Data abstracted included paper type, KF gene and partner, dx, fusion breakpoints, age, stage, MTT received, response to MTT, and CT enrollment. Clinicaltrials.gov was queried for interventional CTs in the U.S. assessing relevant MTTs. We identified 531 studies (299 case reports/series; 119 P-AYA sequencing studies) reporting 4,329 patients with a KF P-AYA solid tumor. Brain tumors had, in decreasing order of frequency, BRAF, NTRK, FGFR, ALK, ROS1, RAF1, MET, and RET fusions. Dx in which KFs were reported were LGG (n=2451), HGG (n=298), embryonal tumors (n=16), ependymomas (n=8), meningioma (n=1), and other (n=16). Extracranial solid tumors (EST) had, in decreasing order of frequency, NTRK, RET, ALK, BRAF, ROS1, MET, FGFR, and RAF1 fusions. Dx in which KFs were reported were thyroid cancer (n=607), sarcoma (n=546), melanoma and spitz tumor (n=183), renal tumor (n=90), carcinoma (n=57), rhabdomyosarcoma (n=12), neuroblastoma (n=11), osteosarcoma (n=9), and other (n=24). As of Aug 23, 2023, 476 CTs were assessing 65 inhibitors relevant for these KFs, of which only 20 are currently FDA-approved. Patients <18 yo were only eligible for 16% of CTs. RET (47%), RAF/BRAF (33%), and NTRK (32%) inhibitors had the highest percent of CTs enrolling patients <18 yo while ERK (4%) and FGFR (6%) inhibitors had the least. Notably, while 467 P-AYA patients identified in our review were <18 yo and had an ALK- or ROS1-fusion positive tumor, only 24% of ALK or ROS1 inhibitor CTs allowed enrollment of patients <18 yo. To our knowledge, this is the first systematic review of KFs in P-AYA solid tumors. Oncogenic KFs are common and occur across P-AYA solid and brain tumor dx demonstrating a need for CTs of relevant MTTs including patients <18 yo, particularly for drugs targeting ALK, ROS1, and FGFR. Citation Format: Lorena Lazo de la Vega, Sebastian K. Eder, Ellen Sukharevsky, Zachary A. Kahn, Michaela O'Connell, Franziska Wachter, Emily P. Anderson, Maria C. Trissal, Jenna J. LaBelle, Kendall Carpenter, Leo Kager, Mariella G. Filbin, Katherine A. Janeway. Perspectives from a systematic review of kinase fusion oncogenes and access to corresponding kinase inhibitor clinical trials in pediatric, adolescent, and young adult solid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2857.

Abstract 4660: Characterization of a nicotinamide-based kinase inhibitor HSN748 for inhibition of RET-driven tumors

Abstract Rearranged during transfection (RET) is a protein tyrosine kinase that is aberrantly activated by gene fusions or mutations in many types of human cancer including thyroid cancer and non-small cell lung cancer (NSCLC). Selpercatinib (LOXO-292) and pralsetinib (BLU-667) are FDA approved RET-selective tyrosine kinase inhibitors (TKIs) being used to treat RET-altered cancer. However, these tumors develop selpercatinib/pralsetinib-resistance via acquisition of RET(G810C/R/S) mutations located at the solvent front of the ATP binding pocket. The overall goal of our research is to discover next-generation of RET kinase inhibitors to inhibit selpercatinib/pralsetinib-resistant RET solvent-front mutants. In this study, we characterized one of the nicotinamide-based RET kinase inhibitor, HSN748, in vitro and in vivo. HSN748 was able to inhibit selpercatinib/pralsetinib-resistant RET(G810C/R) solvent-front mutations. In BaF3/KIF5B-RET(G810C) tumor xenograft experiment, HSN748 treatment resulted in tumor regression. In immune competent transgenic mice, KIF5B-RET-induced lung tumors regressed after HSN748 treatment by oral gavage for one month (10-15 mg/kg, QD) without affecting body weight. These results identify HSN748 as an orally available RET inhibitor capable of inhibiting KIF5B-RET and its solvent-front mutant-driven tumors. Citation Format: Ujjwol Khatri, Neetu Dayal, Tao Shen, Xueqing Hu, Herman Sintim, Jie Wu. Characterization of a nicotinamide-based kinase inhibitor HSN748 for inhibition of RET-driven tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4660.

Abstract 3278: Analysis of the mechanism of resistance to selpercatinib in thyroid cancer cells with RET/PTC1 fusion gene

Abstract Purpose: Precision oncology, in which molecularly targeted drugs are selected based on genetic mutations, has begun in thyroid cancer, and drugs targeting BRAF, RET, and NTRK are already in clinical use. However, as with previous therapeutic agents for malignant tumors, there have been cases of resistance to these drugs either initially or during treatment, and the mechanisms of drug resistance need to be elucidated. So far, little is known about the mechanisms of resistance to molecularly targeted drugs in thyroid cancer. To overcome resistance to the RET inhibitor, selpercatinib (SEL), and to consider next therapeutic strategies, we aimed to elucidate the mechanisms of resistance to SEL. Method: We used papillary carcinoma cell lines TPC-1 and CUTC48, which harbor the RET/PTC1 fusion gene. We established an SEL-resistant subline (TPC-1/SELR) in TPC-1 by long-term exposure to SEL. The growth inhibitory effect of drugs was analyzed using the WST assay. The alteration of intracellular signal transduction was analyzed by western blotting. Result: The 50% inhibitory concentration (IC50) for SEL in the parental TPC-1 was 3 nM, while growth inhibition in TPC-1/SELR did not reach 50% even at more than 100 nM of SEL. On the other hand, growth inhibition by SEL in CUTC48 did not reach 50% even at more than 100 nM of SEL. The sensitivity to SEL in CUTC48 was comparable to that in TPC-1/SELR, and CUTC48 showed an intrinsic resistance to SEL. TPC-1/SELR showed increased phosphorylation of EGFR, Erk, Akt, and mTOR compared to TPC-1. In CUTC48, phosphorylation of EGFR and Erk was not increased compared to that of TPC-1, while phosphorylation of Akt and mTOR was increased. We investigated the growth inhibitory effect of an mTOR inhibitor everolimus and found that TPC-1 (IC50: 1.3 nM), TPC-1/SELR (5 nM), and CUTC48 (9.4 nM) were all highly sensitive to everolimus. Discussion: Our results showed that Akt and mTOR phosphorylation were elevated in the SEL-resistant TPC-1/SELR and the SEL-intrinsic resistant CUTC48 in common, suggesting the involvement of activation of the Akt-mTOR pathway in SEL resistance in thyroid cancer. The inhibition of the Akt-mTOR pathway in parallel with RET may have therapeutic potential to overcome SEL resistance in thyroid cancer. Citation Format: Masatsugu Amitani, Ken-ichi Ito. Analysis of the mechanism of resistance to selpercatinib in thyroid cancer cells with RET/PTC1 fusion gene [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3278.

Abstract 4998: Pralsetinib induces opportunistic infection in RET fusion-positive NSCLC patients via inhibition of IL-2 production by blocking Jak3-Stat5 activation

Abstract Background: Oncogenic alteration in RET, representing 1-2% of non-small cell lung cancer (NSCLC), is one of the important targets. Pralsetinib is a selective RET inhibitor that targets 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 event related to pralsetinib. To investigate the potential impact of pralsetinib on T-cells, we examined cytokine release from Jurkat T (JT) cells after pralsetinib treatment. To test if Stat5 bind to IL-2 promotor, we conducted Chromatin Immunoprecipitation (ChIP). Results: Out of a total of 18 patients with measurable disease, 14 patients (93%) achieved a partial response. With the median follow-up duration of 8.7 months, four cases of opportunistic infections were occurred. Notably, three patients (16.6%) experienced invasive pulmonary aspergillosis, and one patient (5.5%) experienced cytomegalovirus pneumonia. To mimic conditions akin to activated T cells, we treated JT cells with PMA and ionomycin, inducing IL-2 release through the binding of NFAT, AP-1, and NF-κB to the IL-2 promoter. Following approximately two weeks of pralsetinib treatment, a decrease of IL-2 releases, the inhibition of Jak3 and the reduced-expression of JunB and c-Jun was observed. Additionally, to investigate the direct correlation between Jak3 and IL-2 release, JT cells were treated with ritlecitinib, the Jak3-selective inhibitor, for approximately two 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 also showed that the transcription factor Stat5, a direct downstream signaling component of Jak3, binds to the IL-2 promoter during the activation of Jurkat T cells. Conclusion: Pralsetinib inhibits additional IL-2 production by blocking Jak3/Stat5 activation triggered by IL-2 released during early T cell activation. As a result, the Jak3 inhibition induced by pralsetinib leads to a decrease in JunB/c-Jun expression and blocking Stat5 activation by reducing IL-2 release. Consequently, Pralsetinib-related opportunistic infections may be caused by inhibition of the JAK3 pathway and IL-2 release. Citation Format: Shinkyo Yoon, Hyun-Min Ryu, 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 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4998.

Abstract 4238: ModelingRET-rearranged non-small cell lung cancer (NSCLC): Generation of lung cell progenitors from patient-derived induced pluripotent stem cells (iPSCs)

Abstract Rearranged during Transfection (RET) oncogenic rearrangements can occur in 1-2% of lung adenocarcinomas. While RET-driven NSCLC models have been developed using various approaches, no model based on patient-derived induced pluripotent stem cells (iPSCs) has been described yet. Patient-derived iPSCs hold great promise for disease modeling. However, generating iPSCs with specific oncogenic drivers, like RET rearrangements, presents challenges due to reprogramming efficiency and genotypic variability within tumors. To address this issue, we aimed to generate lung progenitor cells (LPCs) from patient-derived iPSCs carrying the somatic mutation RETC634Y which is commonly associated with hereditary medullary thyroid carcinoma. Additionally, we have established a RETC634Y knock-in iPSC model to validate the effect of this oncogenic mutation during LPC differentiation. We successfully generated LPCs from RETC634Y iPSCs using a 16-days protocol and showed an overexpression of cancer-associated markers as compared to control iPSCs. Transcriptomic analysis revealed a distinct signature of NSCLC tumor repression, suggesting a lung multilineage lung dedifferentiation, along with an upregulated signature associated with RETC634Ymutation potentially linked to poor NSCLC prognosis. These findings were validated using the RETC634Y knock-in iPSC model, highlighting key cancerous targets such as PROM2 and C1QTNF6, known to be associated with poor prognostic outcomes. Furthermore, the LPCs derived from RETC634Y iPSCs exhibited a positive response to the RET inhibitor Pralsetinib, evidenced by the downregulation of the cancer markers. This study provides a novel off-the-shelf iPSC model of RET-driven NSCLC, paving the way for exploring the molecular mechanisms involved in RET-driven NSCLC to study disease progression and to uncover potential therapeutic targets. Citation Format: Paul Marcoux, Jinwook Hwang, Christophe Desterke, Jusuf Imeri, Annelise Bennaceur Griscelli, Ali Turhan. ModelingRET-rearranged non-small cell lung cancer (NSCLC): Generation of lung cell progenitors from patient-derived induced pluripotent stem cells (iPSCs) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4238.

Adaptive Darwinian off-target resistance mechanisms to selective RET inhibition in RET driven cancer

Patients treated with RET protein tyrosine kinase inhibitors (TKIs) selpercatinib or pralsetinib develop RET TKI resistance by secondary RET mutations or alterative oncogenes, of which alterative oncogenes pose a greater challenge for disease management because of multiple potential mechanisms and the unclear tolerability of drug combinations. A patient with metastatic medullary thyroid carcinoma (MTC) harboring a RET activation loop D898_E901del mutation was treated with selpercatinib. Molecular alterations were monitored with tissue biopsies and cfDNA during the treatment. The selpercatinib-responsive MTC progressed with an acquired ETV6::NTRK3 fusion, which was controlled by selpercatinib plus the NTRK inhibitor larotrectinib. Subsequently, tumor progressed with an acquired EML4::ALK fusion. Combination of selpercatinib with the dual NTRK/ALK inhibitor entrectinib reduced the tumor burden, which was followed by appearance of NTRK3 solvent-front G623R mutation. Preclinical experiments validated selpercatinib plus larotrectinib or entrectinib inhibited RET/NTRK3 dependent cells, whereas selpercatinib plus entrectinib was necessary to inhibit cells with RET/NTRK3/ALK triple alterations or a mixture of cell population carrying these genetic alterations. Thus, RET-altered MTC adapted to selpercatinib and larotrectinib with acquisition of ETV6::NTRK3 and EML4::ALK oncogenes can be managed by combination of selpercatinib and entrectinib providing proof-of-concept of urgency of incorporating molecular profiling in real-time and personalized N-of-1 care transcending one-size-fits-all approach.

Evaluation of Five-year Overall Survival Rates Among 18,331 Head and Neck Cancer Patients Exposed to Different Targeted Therapies Through Real-world Data in a Case-controlled Study.

Targeted therapy is an important and fast developing aspect of modern tumor therapy including therapy of head and neck cancer (HNC). Surgically treated patients often experience significant limitations to their ability to swallow, speak, or mimic expressions. In cases of recurrent tumors or palliative situations, targeted therapies such as immune checkpoint inhibitors (ICI) are frequently employed. This study compared different targeted therapies focusing on survival probability. Data from patients with head and neck cancer treated with different therapy regimens from the TriNetX network were analyzed. Two groups were formed: Cohort I received one targeted therapy, whereas patients in cohort II received a different targeted therapy. Cohorts I and II were matched 1:1 with respect to certain confounders. After defining the primary outcome as "death", a Kaplan-Meier analysis was performed, and the risk ratio (RR), odds ratio (OR), and hazard ratio (HR) were calculated. A total of 18,331 patients with HNC treated with targeted therapy were analyzed. Patients treated with VEGF inhibitors had a significantly longer overall survival than patients treated with c-MET or EGFR inhibitors. Patients treated with PI3K inhibitors showed a significantly reduced survival probability compared to those treated with c-MET, mTOR, and RET inhibitors. EGFR inhibitors are one of the most frequently used targeted therapies in HNC. However, in the present analysis, a survival advantage of patients treated with c-MET inhibitors or VEGF inhibitors was observed compared to those treated with EGFR inhibitors.

Management of Advanced Medullary Thyroid Carcinoma: Current Systemic Therapy Options.

The current rapid development of more selective and effective drugs for the treatment of thyroid cancer has open a new era in the treatment of patients with this condition, in the past limited to the possibility of only radioactive iodine for well differentiated tumor and surgery for medullary thyroid carcinoma (MTC). The treatment of advanced medullary thyroid carcinoma has evolved in the last few years and options for patients with advanced disease are now available. Multikinase inhibitors (MKIs) with nonselective RET inhibition like Vandetanib and Cabozantinib were approved for the treatment of MTC, although the efficacy is limited due to the lack of specificity resulting in a higher rate of drug-related adverse events, leading to subsequent dose reductions, or discontinuation, and the development of a resistance mechanism like seen on the RET Val804 gatekeeper mutations. MTC is associated with mutations in the RET protooncogene, and new highly selective RET inhibitors have been developed including Selpercatinib and Pralsetinib, drugs that have demonstrate excellent results in clinical trials, and efficacy even in the presence of gatekeeper mutations. However, despite their efficacy and great tolerability, mechanisms of resistance have been described, such as the RET solvent front mutations. Due to this, the need of constant evolution and drug research is necessary to overcome the emergence of resistance mechanisms.

Chinese expert consensus on the diagnosis and treatment of advanced RET fusion-positive non-small cell lung cancer (2023 edition)

Lung cancer is the most common cancer and the leading cause of cancer death in China. Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer. Mutations of driver genes have major impacts on incidence and progression of lung cancer. Advances in molecular biology research and clinical research have promoted the discovery of rare tumor driver genes, as well as the development and application of new targeted drugs. Nearly 1% to 2% of NSCLCs harbor RET fusions, and this patient population may not respond well to traditional treatments like chemotherapy or radiation therapy. After the new highly selective RET inhibitors pralsetinib (BLU-667) and selpercatinib (LOXO-292) entered clinical application, the diagnosis and treatment of RET fusion positive NSCLC has made breakthrough progress. At present, there is a lack of guiding consensus on the standardized diagnosis and treatment of RET fusion-positive NSCLC in China. The Society of Cancer Precision of Chinese Anti-Cancer Association and Lung Cancer Expert Group of Chinese Medical Journal, invited 38 experts form respiratory medicine, medical oncology, oncology radiotherapy and pathology to form a consensus development group. Based on the existing research evidence, combined with China's clinical practice experience, a standardized process for the diagnosis and treatment of advanced RET fusion-positive NSCLC is proposed, including suitable populations and methods for RET gene fusion, treatment drug selection, treatment of resistance to highly selective RET inhibitors, and management of adverse reactions to treatment, with a view to providing guidance for clinicians.

An Overview of the Use of Anti-Angiogenic Agents in the Treatment of Thymic Epithelial Tumors

Angiogenesis significantly influences the carcinogenesis of thymic epithelial tumors (TET). Both thymomas and thymic carcinoma (TC) overexpress VEGF-A and VEGFR-1 and -2. This review aims to provide an appraisal of the use of anti-angiogenics in the treatment of TET. The literature research identified 16 studies that were deemed eligible for further analysis. Seven studies assessed the clinical efficacy of sunitinib and five studies the use of apatinib and/or anlotinib. The multicenter Japanese phase II REMORA trial investigated the efficacy of lenvatinib, which is a multi-targeted inhibitor of VEGFR, FGFR, RET, c-Kit, and other kinases. The objective response rate was 38% (25.6–52%), which is the highest documented in TET that progressed after first-line chemotherapy. Anti-angiogenic agents may be useful in the treatment of TET, which are not amenable to curative treatment. Their toxicity profile seems to be acceptable. However, angiogenesis inhibitors do not appear to have a major influence on either thymomas or TC, although multikinase inhibitors may have some effect on TC. The current evidence suggests that the most active agent is lenvatinib, whereas sunitinib could be proposed as an acceptable second-line therapy for TC. Further research concerning the combination of immune checkpoint inhibitors with anti-angiogenic drugs is warranted.

Abstract B145: Clinical features of progression and outcomes with subsequent therapies in patients treated with RET-inhibitors

Abstract Background: RET fusions can be found in 1-2% of patients (pts) with advanced non-small cell lung cancer (aNSCLC). While targeted treatment with RET inhibitors (RETi) has significantly improved prognosis, resistance eventually develops. Current knowledge regarding the pattern of disease progression and the optimal therapeutic sequence for patients treated with RETi is limited. Methods: We conducted a retrospective multicentric collection of clinical and biological data on pts with RET-rearranged (RET+) aNSCLC treated with first-generation RETi. Patients with a progression-free survival (PFS) period of less than 8 months on RETi were classified as poor responders. We investigated the clinical patterns of disease progression (PD) and outcomes with subsequent therapies, evaluated by the overall response rate (ORR by investigator assessment) and PFS. PFS2 was calculated from RETi start to PD after subsequent line. Results: 173 pts from 32 centres were identified. Median age was 65, 61% were female, 41% were smokers, 81% had an ECOG performance status <2, 95% had adenocarcinoma, 18% had brain metastasis, 76% had RETi in first or second line. The administered RETi were selpercatinib in 55%, pralsertinib in 42%, other first generation RETi in 3% of pts. After a median follow up of 30.4 months (mo) (95%CI 26-32.1), 84 pts (48.6%) had PD after RETi, with a median PFS (mPFS) of 15.6 mo (95%CI 12.2-23.3) and an ORR of 72.2%. Radiological PD patterns included multisite PD in 46 (71%), oligoPD in 15 (23%), mixed response in 4 (6%). 7/15(47%) received local treatment while continuing RETi. Subsequent treatment line after RETi was administered in 37 pts (21%). Considering chemotherapy-based treatments, ORR and mPFS were 46% and 5.52 mo (4.07-11.3) overall, 57% and 4.53 mo (95%CI 1.4-NA) in 9 pts with chemo-immunotherapy, 27% and 5.88 mo (95%CI 2.1-NA) in 11 pts with platinum-doublet, 83% and 10.4 mo (8.28-NR) in 6 pts with bevacizumab plus chemotherapy (p=0.77 vs chemotherapy alone). Targeted therapy was administered in 8 patients: 6 received RETi after RETi (first generation) with 33% ORR and 3.83 mo (1.77-NR) mPFS, one patient had crizotinib plus RETi to target MET amplification found at resistance, with a PFS of 5.95 mo, one had alectinib alone, targeting SQSTM1-ALK fusion found at resistance, with PD after 1.58 mo. Cabozantinib was chosen for one pt, with a PFS of 5.52 mo and obtaining PR; one pt was treated with single-agent immunotherapy, with rapid PD after 1 month; one pt had an investigational antibody-drug conjugate, experiencing SD and a 4 mo PFS. PFS to subsequent treatment was not different between long and poor responders to RETi (13 and 15 pts,5.88 vs 4.53 m, respectively, p=0.75); PFS2 was longer in good responders to RETi (25 vs 16 mo,p=0.012). Conclusions: Disease progression to RETi most frequently involved multiple sites. Chemotherapy plus bevacizumab seemed to perform better than chemotherapy alone or chemo-immunotherapy. The benefit of tailored treatment basing on the molecular profile at PD needs to be further explored. Citation Format: Arianna Marinello, Wael Salem Zrafi, Giacomo Massa, Fabrizio Tabbò, Fabrizio Citarella, Filippo Gustavo Dall'Olio, Lindsay Colin, Emilio Bria, Giulio Metro, Josè Nicolas Minatta, Antonio Calles, Isabelle Monnet, Marco Tagliamento, Hortense Gaultier De Saint Basile, Nicolas Girard, Arianna Pagliaro, Diego Luigi Cortinovis, Judith Raimbourg, Carol Zanchetta, Giannis Mountzios, Frank Aboubakar Nana, Safae Terrisse, Alessandro Russo, Patricia Iranzo, Clarisse Audigier-Valette, Philippe Rochigneux, Vincent Fallet, Michael Duruisseaux, Sophie Cousin, Florian Guisier, Laura Mezquita, Jordi Remon, Anas Gazzah, David Planchard, Benjamin Besse, Mihaela Aldea. Clinical features of progression and outcomes with subsequent therapies in patients treated with RET-inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B145.

Abstract B157: Preclinical evaluation of the blood-brain barrier permeability and intracranial efficacy of the next-generation RET inhibitor Vepafestinib

Abstract Background. Drug resistance and central nervous system (CNS) metastasis often hampers response to precision therapy for RET-rearranged solid tumors. Vepafestinib (Vepa) is a clinical-stage, potent and selective RET tyrosine-kinase inhibitor (TKI) with proven activity against on-target resistance mutations that arise post first-generation RET TKI. Here, we utilized an array of structural and pharmacokinetic methods to determine blood-brain barrier (BBB) permeability of vepa compared with other RET TKIs. Furthermore, we correlate BBB penetrability of RET inhibitors with in vivo efficacy in preclinical models of RET-rearranged intracranial solid tumors. Methods. RET inhibitors were generated by rational chemical design to identify agents which penetrate BBB more effectively than currently approved RET TKIs, and with reduced efflux transporter susceptibility. Transcellular transport assays were conducted in LLC-PK1 and MDCK II cells expressing P-gp and BCRP, respectively. Brain penetrability was assessed by total (Kp) and unbound (Kp,uu) brain:plasma concentration ratios in male Balb/c mice and microdialysis studies in freely-moving male Wistar rats. Models of CNS metastasis were generated by implanting cells labelled with luciferase into the cerebellum of mice. Studies were conducted in comparison with selpercatinib (Selp), pralsetinib (Pral) and TPX-0046. Results. Assessment of Kp and Kp,uu parameters of analogs revealed an excellent structure-activity relationship where modification at the 6-position of the pyrrolopyrimidine core resulted in improved BBB penetrability. The analog with the most favorable physiochemical and biological properties was Vepa. Vepa showed poor susceptibility to P-gp and BCRP-mediated efflux and better CNS penetrability (Kp,uu: 1.3) and retention than Sel, Pral and TPX-0046. The ratio of the observed concentrations of Vepa in microdialysates from the prefrontal cortex, cerebrospinal fluid (CSF) and plasma free fraction was approximately 1:1:1; these ratios were maintained from 2h to 6.5h after Vepaadministration (up to 8h for CSF). Importantly, Vepa was more effective than Selp at causing regression of intracranial xenograft tumors of RET fusion-driven malignancies (lung adenocarcinoma and sarcoma), thus extending survival of tumor-bearing animals. Common first-generation RET TKI resistance mutations at the solvent front (G810A/C/S/R), gatekeeper (V804L/M), hinge (Y806C) and roof (L730Q/R) regions remained sensitive to Vepa. Vepa was more selective than Selp, Pral and TPX-0046, inhibiting only RET when profiled against a panel of 256 kinases. In contrast, Selp, Pral and TPX-0045 also inhibited VEGFR2, and 4, 11 and 39 additional kinases, respectively. Conclusions. Vepafestinib is a structurally distinct and specific RET inhibitor, with superior brain penetration and retention kinetics. It is more effective than selpercatinib in controlling CNS disease. Vepafestinib is currently undergoing phase 1 and 2 clinical trial for patients with advanced solid tumors with RET alterations (margaRET, NCT04683250). Citation Format: Igor Odintsov, Kentaro Wakayama, Tom Zhang, Satoru Iguchi, Masanori Kato, Allan JW Lui, Inna Khodos, Morana Vojnic, Claudio Giuliano, Annalisa Bonifacio, Monika A Davare, Elisa de Stanchina, Emanuela Lovati, Marc Ladanyi, Isao Miyazaki, Romel Somwar. Preclinical evaluation of the blood-brain barrier permeability and intracranial efficacy of the next-generation RET inhibitor Vepafestinib [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B157.

Abstract B043: Preliminary results from a phase I/II study evaluating the safety, tolerability, and efficacy of EP0031, a next generation selective RET inhibitor, in patients with advanced RET-altered malignancies

Abstract BACKGROUND Emergence of resistance to 1st generation (gen) selective RET inhibitors (SRIs, selpercatinib/pralsetinib) is an area of high unmet need. EP0031 (A400/KL590586) has promise as a next gen SRI. Clinical data was recently reported from an ongoing Phase I/II study in China that included 109 patients (pts) with RET-altered tumours (NCT05265091, Zhou et al. JCO 2023, 41:16, suppl. 3007). ORR was 80.8 and 69.7%, across 40-120 mg QD doses, in treatment-naïve or pre-treated metastatic NSCLC, respectively. Responses durable with longest 14.5 mo ongoing, median DoR not reached. Responses, including in the CNS, observed in 5/7 pts that previously received 1st gen SRI. EP0031 had an encouraging safety and tolerability profile. 90mg QD was selected as RP2D. Here we report initial data from the dose ranging and optimisation module of a parallel Phase I/II study in the US and Europe. EXPERIMENTAL PROCEDURES This study (NCT05443126) is recruiting pts with RET-altered NSCLC, thyroid cancer or other solid tumours. Study includes male or female pts ≥ 18 years of age, PS 0 or 1 with/without asymptomatic, stable brain metastases. The first module is a dose escalation to investigate safety, tolerability, PK/PD and define the MTD and/or RP2D. This is based on rolling 6 design and is expected to recruit up to 40 pts. RESULTS As of 10th July 2023, a total of 15 pts, 5 medullary thyroid cancer (MTC), 2 CRC and 8 NSCLC, have been included across dose cohorts of 20mg (N=3), 60mg (N=6) and 90mg (N=6). PK profile is comparable with Chinese pts and consistent with QD dosing. 60mg QD predicted to achieve IC50 for inhibition of RET fusions/mutations. No DLTs and no serious adverse events reported. No dose reductions, interruptions or discontinuations have been required. AEs have been mainly Grade 1 or 2. No neutropenia, hypertension, neurotoxicity or QTc prolongation observed. Three pts (all MTC) that received 20 mg QD had their dose escalated to 60mg (after 5-7 cycles at 20mg). 2 of those pts, both SRI naïve, have ongoing stable disease for >23 weeks. 1 pt (prior SRI) with a new target lesion continues to derive clinical benefit and remains on treatment. Of 4 evaluable pts at 60 mg, 2 pts with NSCLC (prior SRI) have achieved a PR, treatment is ongoing and 2 pts with MTC (prior SRI) had PD, one of whom continues to derive clinical benefit and remains on treatment. The remaining pts at 60 or 90mg (potential RP2D) are not yet evaluable for efficacy. Genomic analyses for RET/non-RET resistance mechanisms are ongoing. Expansion of the 60mg and 90mg cohorts is planned for dose optimization. Expanded efficacy, safety and PK/PD data from dose ranging and optimization cohorts will be presented. CONCLUSIONS These preliminary data are encouraging, consistent with that reported from a larger cohort of Chinese patients, with early evidence that EP0031 is active in pts that received prior 1st gen SRI. Once RP2D confirmed, the study will expand cohorts of both SRI-naïve and pre-treated pts with RET-altered NSCLC, thyroid and other solid tumours. Citation Format: Elena Garralda, Alonso Guzman, Pilar Garrido, Andrew G Gianoukakis, Matthew Taylor, Sarina Piha-Paul, Matthew G Krebs, Antoine Italiano, Liz Clark, Geoff Fisher, Tobi Arkenau, Vivek Subbiah. Preliminary results from a phase I/II study evaluating the safety, tolerability, and efficacy of EP0031, a next generation selective RET inhibitor, in patients with advanced RET-altered malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B043.

Can New Structure of RET Inhibitors Capable of Suppressing Resistant in Non-Small-Cell Lung Cancer?

In 2012, RET rearrangements are observed in 1-2% of Non-Small-Cell Lung Cancer (NSCLC) patients and result in the constitutive activation of downstream pathways normally implied in cell proliferation, growth, differentiation and survival. Several compounds have been reported, including some traditional kinases inhibitors and the discovery of some new structure of natural products. Cabozantinib and vandetanib are multikinase inhibitors have been explored in the clinic for NSCLC patients. As a result of the nonselective nature of these multikinase inhibitors, patients had off-target adverse effects. Then, the discovery and clinical validation of highly potent selective RET inhibitors such as pralsetinib and selpercatinib demonstrating improved effificacy and a more favorable toxicity profile. However, acquired resistance mediated by secondary mutations in the solvent-front region of the kinase (e.g. G810C/S/R) becomes a major challenge for selective RET inhibitor therapies. In this review, we will highlight typical RET inhibitors developed during these years and provide a reference for more potential RET inhibitors exploration in the future.

Clinical Outcomes of Radioactive Iodine Redifferentiation Therapy in Previously Iodine Refractory Differentiated Thyroid Cancers.

Objective: Redifferentiation therapy (RDT) can restore radioactive iodine (RAI) uptake in differentiated thyroid cancer (DTC) cells to enable salvage 131I therapy for previously RAI refractory (RAIR) disease. This study evaluated the clinical outcomes of patients who underwent RDT and identified clinicopathologic characteristics predictive of RAI restoration following RDT. Methods: This is a retrospective case series of 33 patients with response evaluation criteria in solid tumors (RECIST)-progressive metastatic RAIR-DTC who underwent RDT between 2017 and 2022 at the Mayo Clinic (Rochester, MN). All patients underwent genomic profiling and received MEK, RET or ALK inhibitors alone, or combination BRAF-MEK inhibitors for 4 weeks. At week 3, those with increased RAI avidity in metastatic foci received high-dose 131I therapy. Baseline and clinicopathologic outcomes were comprehensively reviewed. Results: Of the 33 patients, 57.6% had restored RAI uptake following RDT (Redifferentiated subgroup). 42.1% (8/19) with papillary thyroid cancers (PTC), 100% (4/4) with invasive encapsulated follicular variant PTCs (IEFV-PTCs), and 100% (7/7) with follicular thyroid cancers (FTC) redifferentiated. All (11/11) RAS mutant tumors redifferentiated compared with 38.9% (7/18) with BRAF mutant disease (6 PTC and 1 IEFV-PTC). 76.5% (13/17) of redifferentiated and 66.7% (8/12) of non-redifferentiated patients achieved a best overall RECIST response of stable disease (SD) or non-complete response/non-progressive disease. Both subgroups had a median 12% tumor shrinkage at 3 weeks on drug(s) alone. The redifferentiated subgroup, following high-dose 131I therapy, achieved an additional median 20% tumor reduction at 6 months after RDT. There were no statistically significant differences between both groups in progression free survival (PFS), time to initiation of systemic therapy, and time to any additional therapy. Of the entire cohort, 6.1% (2/33) experienced histologic transformation to anaplastic thyroid cancer, 15.1% (5/33) died, and all had redifferentiated following RDT and received 131I therapy. Conclusion: RDT has the potential to restore RAI avidity and induce RECIST responses following 131I therapy in select patients with RAIR-DTC, particularly those with RAS-driven "follicular" phenotypes. In patients with PTC, none of the evaluated clinical outcomes differed statistically between the redifferentiated and non-redifferentiated subgroups. Further studies are needed to better characterize the long-term survival and/or safety outcomes of high-dose RAI following RDT, particularly whether it could be associated with histologic anaplastic transformation.

RET inhibitors in thyroid cancer: A single-institution experience

RET inhibitors in thyroid cancer: A single-institution experience

Design and synthesis of indol-2-one derivatives as potential RET inhibitors

We synthesized and assessed five series of indol-2-one derivatives for their potential as RET kinase inhibitors. Notably, compounds B3, B6, D1, D2, D3, and D5 demonstrated significant inhibitory activity. Among these, D5 exhibited the best activity of inhibiting RET kinase, which provided reference for the subsequent development of RET kinase inhibitors as anti-thyroid cancer chemical.