RET Phosphorylation Research Articles

Mesenchymal cells regulate enteric neural crest cell migration via RET-GFRA1b trans-signaling

During early development, the enteric nervous system forms from the migration of enteric neural crest cells (ENCCs) from the foregut to the hindgut, where they undergo proliferation and differentiation facilitated by interactions with enteric mesenchymal cells (EMCs). This study investigates the impact on ENCC migration of EMC-ENCC communication mediated by GFRA1b expressed in EMCs. GFRA1-expressing cells in day 11–12 (E11–12) mouse embryos differentiated into smooth muscle cells from E12 onwards. Observations at E12–13.5 revealed high levels of GFRA1 expression on the anti-mesenteric side of the hindgut, correlating with enhanced ENCC migration. This indicates that GFRA1 in EMCs plays a role in ENCC migration during development. Examining GFRA1 isoforms, we found high levels of GFRA1b, which lacks amino acids 140–144, in EMCs. To assess the impact of GFRA1 isoforms on EMC-ENCC communication, we conducted neurosphere drop assays. This revealed that GFRA1b-expressing cells promoted GDNF-dependent extension and increased neurite density in ENCC neurospheres. Co-culture of ENCC mimetic cells expressing RET and GFRA1a with EMC mimetic cells expressing GFRA1a, GFRA1b, or vector alone showed that only GFRA1b-expressing co-cultured cells sustained RET phosphorylation in ENCC-mimetic cells for over 120 min upon GDNF stimulation. Our study provides evidence that GFRA1b-mediated cell-to-cell communication plays a critical role in ENCC motility in enteric nervous system development. These findings contribute to understanding the cellular interactions and signaling mechanisms that underlie enteric nervous system formation and highlight potential therapeutic targets for gastrointestinal motility disorders.

Abstract LB330: FHND5071: a selective RET inhibitor with unique pharmacokinetic profile

Abstract Background: Gene rearrangements (fusions) and mutations in RET have the potential to be oncogenic drivers and have been observed in a variety of tumors types. Selective RET inhibitors selpercatinib and pralsetinib have been approved for patients with RET-altered cancers. FHND5071 is a novel kinase inhibitor which specifically targets RET activated forms and has unique pharmacokinetic profile. Methods: The pharmacological profile of FHND5071 have been confirmed in in vitro and in vivo evaluations, including enzyme and cell-based assays, PK/PD study, and RET-dependent tumor models. Results: In enzymatic assays, FHND5071 potently inhibited RET wild type, RET fusions and mutations with the IC50 of 4.47~19.26 nM and demonstrated 89-fold selectivity over KDR (VEGFR-2). FHND5071-M2, the active metabolite identified in pre-clinical pharmacokinetics, had the same potency as FHND5071. In cellular settings, FHND5071 inhibited the proliferation of Ba/F3 engineered cells (RET WT, RET V804M, RET M918T) and inhibited RET phosphorylation in HEK-293 engineered cells (WT, V804M, RET M918T, KIF5B-RET, CCDC6-RET) with the similar potency compared with selpercatinib. In PK/PD study (Ba/F3 KIF5B-RET allograft model), compared with selpercatinib, FHND5071 had longer Tmax and MRT in plasma and tumor tissue. FHND5071 preferred to be distributed into tissues. The exposure of FHND5071 in tumor tissue (AUC0-t) was ~28 times that of selpercatinib and the concentration of FHND5071 in brain tissue was ~33 times that of selpercatinib at 4 h after dosing. Meanwhile, after FHND5071 administration, RET phosphorylation and the downstream signaling protein (pAKT, pErk1/2) were significantly inhibited in tumor tissue and the inhibition was maintained for 24 h at least. But the duration of inhibition with selpercatinib was shorter than that of FHND5071. In tumor models, FHND5071 exhibited significant anti-tumor efficacy in Ba/F3 KIF5B-RET model at oral dose levels ≥3 mg/kg once daily (QD) without inducing significant toxicity. FHND5071 QD exhibited similar anti-tumor activity as selpercatinib administrated twice a day (BID) at 30 mg/kg. 30 mg/kg FHND5071 (QD) demonstrated significant anti-tumor efficacy with 100% TGI in patient-derived xenograft models (colorectal cancer with CCDC6-RET and ovarian cancer with NCOA4-RET). In CCDC6-RET intracranial xenograft model, 30 mg/kg FHND5071 (QD) significant prolonged life time of model mice. The median survival time was 56 days and the increase in life-span was 51% (p<0.005 compared with the vehicle group). Conclusions: FHND5071 demonstrated excellent selectivity and anti-tumor efficacy. The unique pharmacokinetic profile of FHND5071 supported the frequency of once-a-day dosing and better efficacy in target organ and brain tumor in clinical. FHND5071 has received IND approval from NMPA and FDA. It is currently in the dose-escalation segment of a first-in-human phase 1 trial for patients with RET-driven solid tumors. Citation Format: Yongqiang Zhu, Jia Wang, Minghong Shang. FHND5071: a selective RET inhibitor with unique pharmacokinetic profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB330.

Abstract 5363: The preclinical selectivity and activity of APS03118, a highly selective and potent next-generation RET inhibitor

Abstract Backgroud: Oncogenic RET is an actionable target across a variety of cancers. Selective RET inhibitors selpercatinib and pralsetinib were recently approved by the FDA and EMA for patients with RET-dependent NSCLC and thyroid cancers. The solvent front mutations (SFMs) RET G810C/S/R have been identified as mechanisms of acquired resistance to both drugs. The gatekeeper RET V804 mutation cannot be inhibited by selected next-generation RET inhibitors in development. APS03118 is a novel next-generation RET inhibitor which is potent against a range of RET fusions and mutations including both SFMs and gatekeeper mutations. Methods: The selectivity, anti-RET activity, and intracranial efficacy of APS03118 were confirmed in vitro and in vivo in a variety of RET-dependent tumor models. Results: APS03118 was highly selective against a panel of 468 kinases and demonstrated 130-fold selectivity over VEGFR-2. In enzymatic assays, APS03118 showed low nanomolar potency against wild type RET and 25 RET mutations/fusions, including the inhibition of RET G810R/C/S (IC50 0.04-5 nM) and RET V804M/L/E (IC50 0.04-1 nM). APS03118 inhibited RET phosphorylation (IC50 <15 nM) in Ba/F3 engineered RET cells (WT, G810R, V804M, M918T). In cell proliferation assays, APS03118 potently inhibited KIF5B-RET Ba/F3 (WT, V804M, V804L, M918T), CCDC6-RET Ba/F3 (WT, V804M, S904F), LC2/ad (CCDC6-RET), TT (RET C634W) (IC50 < 10nM); Ba/F3 RET G810R and G810S IC50 (8-65 nM). APS03118 demonstrated marked anti-tumor efficacy in vivo in RET-driven cell-derived (Ba/F3 KIF5B-RET, V804M, TT (C634W)) and patient-derived (KIF5B-RET, CCDC6-RET, CCDC6-RET V804M) xenograft tumor models. At 10 mg/kg BID, tumor regression was observed in these xenograft models (TGI 87-108%). Tumors completely subsided in CCDC6-RET orthotopic brain model with a 100% survival rate. In the Ba/F3 KIF5B-RET G810R xenograft model, APS03118 30 mg/kg BID showed 90% TGI and was well tolerated, and RET G810 mutations often drive clinical progression on current RET inhibitors. Conclusions: APS03118 is a novel highly selective next-generation RET inhibitor that possesses potent in vitro and in vivo activity against a diverse range of RET alterations, including SFMs-mediated resistance. A first-in-human phase 1 trial for patients with RET-driven solid tumors with activating RET alterations is planned for 2022. Citation Format: Alexander Drilon, Jun Zhong, Ying Lu, Yongbo Liu, Hao Wang, Minchun Chen, Xiaohu Chen, John Zhu, Shun Lu, Vivek Subbiah. The preclinical selectivity and activity of APS03118, a highly selective and potent next-generation RET inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5363.

The development of APS03118, a potent next-generation RET inhibitor for treating RET-inhibitor-resistant patients.

e15107 Background: Oncogenic RET is an actionable target across a variety of cancers. Selective RET inhibitors selpercatinib and pralsetinib were recently approved by the FDA and EMA for patients with RET-dependent NSCLC and thyroid cancers. The solvent front mutations (SFMs) RET G810C/S/R have been identified as mechanisms of acquired resistance to both drugs. APS03118 is a novel next-generation RET inhibitor which is potent against a range of RET fusions and mutations including both SFMs and gatekeeper mutations. Methods: The selectivity, anti-RET activity, and intracranial efficacy of APS03118 were confirmed in vitro and in vivo in a variety of RET-dependent tumor models. Results: APS03118 was highly selective against a panel of 468 kinases and demonstrated 130-fold selectivity over VEGFR-2. In enzymatic assays, APS03118 showed low nanomolar potency against wild type RET and 25 RET mutations/fusions, including the inhibition of RET G810R/C/S (IC50 0.04-5 nM) and RET V804M/L/E (IC50 0.04-1 nM). APS03118 inhibited RET phosphorylation (IC50 < 15 nM) in Ba/F3 engineered RET cells (WT, G810R, V804M, M918T). In cell proliferation assays, APS03118 potently inhibited the proliferation of KIF5B-RET Ba/F3 (WT, V804M, V804L, M918T), CCDC6-RET Ba/F3 (WT, V804M, S904F), LC2/ad (CCDC6-RET), TT (RET C634W) (IC50 < 10nM); Ba/F3 RET G810R and G810S IC50 (8-65 nM). APS03118 demonstrated marked anti-tumor efficacy in vivo in RET-driven cell-derived (Ba/F3 KIF5B-RET, V804M, TT (C634W)) and patient-derived (KIF5B-RET, CCDC6-RET, CCDC6-RET V804M) xenograft tumor models at 10 mg/kg (TGI 87-108%). Tumors completely subsided in CCDC6-RET orthotopic brain model with a 100% survival rate. In the Ba/F3 KIF5B-RET G810R xenograft model, APS03118 30 mg/kg showed 90% TGI and was well tolerated, and RET G810 mutations often drive clinical progression on current RET inhibitors. The pharmacokinetic/pharmacodynamic relationship of APS03118 in Ba/F3 KIF5B-RET G810R and WT xenograft tumor model was investigated, and the sustained decrease of the phosphorylated RET were observed at 30 mg/kg with the plasma exposure exceed cell IC90. Conclusions: APS03118 is a novel highly selective next-generation RET inhibitor that possesses potent in vitro and in vivo activity against a diverse range of RET alterations, including SFMs-mediated resistance. APS03118 has received IND approval and Fast Track Designation from FDA, and a first-in-human phase 1 trial for patients with RET-driven solid tumors with activating RET alterations is planned for 2022.

Abstract P233: TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations

Abstract Fusions involving RET receptor tyrosine kinase are a common driver of tumors across different tissue types, such as lung, thyroid, colorectal, soft tissue and others. TAS0953/HM06 (hereby referred to as HM06) is a novel 2ndgeneration RET-specific inhibitor that is effective against RET solvent front (G810) and gatekeeper (V804) mutations. Here, we evaluated the efficacy of HM06 in lung and thyroid carcinomas, and soft-tissue sarcoma cell lines and PDXs derived from RET inhibitor-naive tumor samples or from tumors with acquired resistance to selpercatinib. HM06 was more effective than the RET multi-kinase inhibitors cabozantinib and vandetanib, and as effective as selpercatinib and pralsetinib in inhibiting growth of patient-derived and isogenic lung, thyroid and sarcoma cell lines (IC50=0.02-0.1 µM) harboring different RET fusions (KIF5B-RET, CCDC6-RET, TRIM33-RET, SPECCL1-RET) or activating mutations (RET C634W). Growth of non-tumor cells was up to 80-fold less sensitive to HM06 (IC50= 1.6 µM). Treatment of RET fusion-positive lung cancer cells with HM06 resulted in a dose-dependent inhibition of RET phosphorylation (Y905 and Y1062) and the downstream effectors AKT, ERK1/2, p70S6K and S6. Caspase 3/7 activity and markers of apoptosis (BIM, cleaved PARP) were induced by HM06 to a similar extent as pralsetinib and selpercatinib (dose range: 0.05-1 µM). HM06 induced changes in the core mediators of cell cycle regulation (upregulation of p27, downregulation of CCND1) and suppressed expression of MYC and ETV5. In vivo, HM06 blocked tumor growth and/or induced regression of up to 65% in seven patient-derived xenograft (PDX) models with RET fusions (five NSCLC PDXs, one sarcoma PDX and one NSCLC cell-line xenograft) to a similar extent as pralsetinib and selpercatinib. However, 6 weeks after cessation of treatment of the SPECCL1-RET-driven sarcoma PDX model, growth of tumors treated with HM06 was suppressed completely, whereas 3/5 pralsetinib-treated tumors and 1/5 selpercatinib-treated tumor regrew. Combination of HM06 and the MET inhibitor capmatinib effectively blocked growth of PDX tumors in a model that was derived from a patient sample that expressed RET fusion and METamplification, and was resistant to selpercatinib. These results suggest that HM06 may be an effective therapy for RET-driven tumors in a tissue-type agnostic manner and can effectively address common on-target and off-target resistance mechanisms such as RET G810X and V804X mutations. HM06 is currently in a phase 1 and 2 clinical trial for patients with advanced solid tumors with RET gene abnormalities (margaRET, NCT 04683250). Citation Format: Igor Odintsov, Renate I. Kurth, Kota Ishizawa, Lukas Delasos, Allan J.W. Lui, Inna Khodos, Connor J. Hagen, Qing Chang, Marissa S. Mattar, Morana Vojnic, Siddharth Kunte, Annalisa Bonifacio, Claudio Giuliano, Elisa De Stanchina, Emily Cheng, Emanuela Lovati, Marc Ladanyi, Romel Somwar. TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P233.

Abstract 2864: RET as a novel therapeutic target in breast cancer brain metastases

Abstract Brain metastases (BrM) are reported in approximately 10-30% of breast cancer patients with a poor median overall survival of 8.5 months. Current therapeutic strategies are mostly limited to surgery, chemotherapy and radiotherapy, making the study of novel druggable targets and brain-permeable small molecule inhibitors an urgent clinical need. Our lab recently reported a hybrid-capture exome RNA-sequencing study on 21 patient-matched pairs of primary breast tumors and brain metastases and identified upregulation of several receptor tyrosine kinases including RET in BrM. The ligand for RET is Glial Cell Derived Neurotrophic Factor (GDNF), which is released by astrocytes and microglia in response to neuroinflammation and brain tissue damage. Clinical BrM cell growth in ex vivo cultures was reduced by the multikinase inhibitor Cabozantinib, indicating a potential role for RET in the growth of BrM lesions. We hypothesize that the high level of brain-derived GDNF provides a permissive microenvironment for RET overexpressing breast cancer cells to form brain metastases, and will evaluate the therapeutic potential of RET targeted therapies for breast cancer BrM patients. Our preliminary data show that in RET-overexpressing (RET-OE) breast cancer cell models, 30-minute GDNF treatment induces RET phosphorylation at two distinct tyrosine residues, along with downstream signaling illustrated by ERK1/2, AKT, and STAT3 phosphorylation. This induction was effectively abrogated after shRNA-mediated RET knockdown. We employed a phospho-kinase array to identify novel downstream targets regulated by the GDNF-RET signaling axis, and found that GDNF induces phosphorylation of multiple targets (including p27 and RSK) in RET-OE cells. Interestingly, phosphorylation of multiple Src family kinases (Lck, Lyn, Fgr) was downregulated with GDNF treatment. We will further examine these downstream targets via reverse phase protein array (RPPA). GDNF triggers migration of RET-OE cell lines in both 2D wound scratch and 3D transwell migration assays. Additionally, we present results obtained through an ex vivo organotypic coculture system, in which RET-OE cells are cultured on top of 300-micron thick murine brain, kidney, or liver slices. RET overexpression increases colony formation compared with empty vector control, suggesting a role for RET signaling in the initial establishment of metastatic brain lesions. Differences in colonization between empty vector transfected and RET-OE cells remain minimal in kidney and liver sections, suggesting a role for RET specific to the brain microenvironment. We will examine RET-driven brain metastasis in vivo by employing RET-OE and RET-deficient cell lines in intracranial, intracardiac, and mammary fat pad injection. These models of BrM will be randomized to RET inhibitor or vehicle treatment to evaluate the efficacy of RET-targeted therapy in the setting of breast cancer brain metastasis. Citation Format: Simeng Liu, Geoffrey Pecar, Jagmohan Hooda, Jennifer M. Atkinson, Fangyuan Chen, Adrian V. Lee, Steffi Oesterreich. RET as a novel therapeutic target in breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2864.

RET Regulates Human Medullary Thyroid Cancer Cell Proliferation through CDK5 and STAT3 Activation.

Medullary thyroid cancer (MTC) is a neuroendocrine tumor that arises from the parafollicular C-cells, which produces the hormone calcitonin. RET is a transmembrane receptor protein-tyrosine kinase, which is highly expressed in MTC. Our previous studies reported that cyclin-dependent kinase 5 (CDK5) plays a crucial role in cancer progression, including MTC. However, the role of CDK5 in GDNF-induced RET signaling in medullary thyroid cancer proliferation remains unknown. Here, we investigated RET activation and its biochemically interaction with CDK5 in GDNF-induced medullary thyroid cancer proliferation. Our results demonstrated that GDNF stimulated RET phosphorylation and thus subsequently resulted in CDK5 activation by its phosphorylation. Activated CDK5 further caused STAT3 activation by its specific phosphorylation at Ser727. Moreover, we also found that GDNF treatment enhanced ERK1/2 and EGR1 activity, which is involved in p35 activation. Interestingly, we identified for the first time that CDK5 physically interacted with RET protein in MTC. Overall, our results provide a new mechanism for medullary thyroid cancer cell proliferation, suggesting that targeting CDK5 may be a promising therapeutic candidate for human medullary thyroid cancer in the near future.

Loss of RET Promotes Mesenchymal Identity in Neuroblastoma Cells.

Simple SummaryThe anaplastic lymphoma kinase (ALK) and rearranged during transfection (RET) receptor tyrosine kinases (RTKs) are expressed in both the developing neural crest and the pediatric cancer neuroblastoma. Moreover, ALK is mutated in approximately 10% of neuroblastomas. Here, we investigated ALK and RET in neuroblastoma, with the aim of better understanding their respective contributions. Using neuroblastoma cell lines, we show that ALK modulates RET signaling at the level of RET phosphorylation, as well as at the level of transcription. Using CRISPR/Cas9, we generated RET knockout neuroblastoma cell lines and performed a multi-omics approach, combining RNA-Seq and proteomics to characterize the effect of deleting RET in a neuroblastoma context. Remarkably, we could show that loss of RET results in a striking epithelial-to-mesenchymal transition (EMT) phenotype, and we provide evidence that RET activity suppresses the mesenchymal phenotype in neuroblastoma.Aberrant activation of anaplastic lymphoma kinase (ALK) drives neuroblastoma (NB). Previous work identified the RET receptor tyrosine kinase (RTK) as a downstream target of ALK activity in NB models. We show here that ALK activation in response to ALKAL2 ligand results in the rapid phosphorylation of RET in NB cells, providing additional insight into the contribution of RET to the ALK-driven gene signature in NB. To further address the role of RET in NB, RET knockout (KO) SK-N-AS cells were generated by CRISPR/Cas9 genome engineering. Gene expression analysis of RET KO NB cells identified a reprogramming of NB cells to a mesenchymal (MES) phenotype that was characterized by increased migration and upregulation of the AXL and MNNG HOS transforming gene (MET) RTKs, as well as integrins and extracellular matrix components. Strikingly, the upregulation of AXL in the absence of RET reflects the development timeline observed in the neural crest as progenitor cells undergo differentiation during embryonic development. Together, these findings suggest that a MES phenotype is promoted in mesenchymal NB cells in the absence of RET, reflective of a less differentiated developmental status.

Artemin Is Upregulated by TrkB Agonist and Protects the Immature Retina Against Hypoxic-Ischemic Injury by Suppressing Neuroinflammation and Astrogliosis.

Hypoxic-ischemia (HI) is a major cause of acquired visual impairment in children from developed countries. Previous studies have shown that systemic administration of 7,8-dihydroxyavone (DHF), a selective tropomyosin receptor kinase B (TrkB) agonist, provides long-term neuroprotection against HI injury in an immature retina. However, the target genes and the mechanisms of the neuroprotective effects of TrkB signaling are not known. In the present study, we induced an HI retinal injury through unilateral common carotid artery ligation followed by 8% oxygen for 2 h in P7 rat pups. DHF was administered intraperitoneally 2 h before and 18 h after the HI injury. A polymerase chain reaction (PCR) array was used to identify the target genes upregulated after the DHF treatment, which was then confirmed with quantitative real-time reverse transcriptase PCR and a western blot. Effects of the downstream mediator of DHF were assessed using an intravitreal injection of neutralizing antibody 4 h after DHF administration (24 h after HI). Meanwhile, the target protein was injected into the vitreous 24 h after HI to validate its protective effect when exogenously supplemented. We found that systemic DHF treatment after HI significantly increased the expression of the artemin (ARTN) gene and protein at P8 and P10, respectively. The neuroprotective effects of DHF were inhibited after the ARTN protein blockade, with an increase in neuroinflammation and astrogliosis. ARTN treatment showed long-term protection against HI injury at both the histopathological and functional levels. The neuroprotective effects of ARTN were related to a decrease in microglial activation at P17 and attenuation of astrogliosis at P29. ARTN enhances phosphorylation of RET, ERK, and JNK, but not AKT or p38 in the immature retina. Altogether, these results suggest that the neuroprotective effect of a TrkB agonist is partially exerted through a mechanism that involves ARTN because the protective effect is ameliorated by ARTN sequestration. ARTN treatment after HI injury protects the immature retina by attenuating late neuroinflammation and astrogliosis in the immature retina relating to the ARTN/RET/JNK/ERK signaling pathway. ARTN may be a strategy by which to provide long-term protection in the immature retina against HI injury.

The next-generation RET inhibitor TPX-0046 is active in drug-resistant and naïve RET-driven cancer models.

3616 Background: RET fusions/mutations drive oncogenesis in lung and thyroid cancers, and several other malignancies. Selective RET inhibitors (selpercatinib/pralsetinib) are active in patients with these cancers; unfortunately, resistance often occurs. On-target resistance includes the acquisition of solvent front mutations (SFMs i.e. RET G810 substitutions). TPX-0046 is a structurally differentiated RET inhibitor that is potent against a range of RET fusions and mutations including SFMs. Methods: The rationally-designed, compact, macrocyclic RET/SRC inhibitor TPX-0046 was characterized in RET-driven in vitro and in vivo tumor models. Results: In enzymatic assays, TPX-0046 showed low nanomolar potency against wild-type RET and 18 RET mutations/fusions. It was potent against SRC and spared VEGFR2/KDR. TPX-0046 inhibited RET phosphorylation (IC50 < 10 nM) in tumor cell lines (LC2/ad, CCDC6-RET; TT, RET C634W) and Ba/F3 engineered RET models (WT, G810R). In cell proliferation assays, TPX-0046 inhibited KIF5B-RET Ba/F3, LC2/ad, and TT cells with IC50 values ~1 nM. Ba/F3 RET engineered cells with SFMs (e.g. G810C/R/S) were potently inhibited by TPX-0046 (mean proliferation IC50 1–17 nM). TPX-0046 demonstrated marked in vivo anti-tumor efficacy in RET-driven cell-derived and patient-derived xenograft tumor models. In a Ba/F3 KIF5B-RET xenograft model, a single dose of 5 mg/kg TPX-0046 inhibited > 80% of RET phosphorylation (corresponding mean free plasma concentration: 51 nM). At 5 mg/kg BID, tumor regression was observed in RET-dependent xenograft models, including those that harbor RET SFMs: TT, CTG-0838 PDX (NSCLC, KIF5B-RET), CR1520 PDX (CRC, NCOA4-RET), Ba/F3 KIF5B-RET, and Ba/F3 KIF5B-RET G810R. Conclusions: TPX-0046 is a unique next-generation RET inhibitor that possesses potent in vitro and in vivo activity against a diverse range of RET alterations, including SFM-mediated resistance. A phase 1/2 trial for RET inhibitor-resistant and naïve RET-driven cancers is on-going (NCT04161391).

RET receptor expression and interaction with TRK receptors in neuroblastomas.

Neuroblastomas (NBs) have heterogeneous clinical behavior, from spontaneous regression or differentiation to relentless progression. Evidence from our laboratory and others suggests that neurotrophin receptors contribute to these disparate behaviors. Previously, the role of TRK receptors in NB pathogenesis was investigated. In the present study, the expression of RET and its co‑receptors in a panel of NB cell lines was investigated and responses to cognate ligands GDNF, NRTN, and ARTN with GFRα1‑3 co‑receptor expression, respectively were found to be correlated. RET expression was high in NBLS, moderate in SY5Y, low/absent in NBEBc1 and NLF cells. All cell lines expressed at least one of GFRα co‑receptors. In addition, NBLS, SY5Y, NBEBc1 and NLF cells showed different morphological changes in response to ligands. As expected, activation of RET/GFRα3 by ARTN resulted in RET phosphorylation. Interestingly, activation of TrkA by its cognate ligand NGF resulted in RET phosphorylation at Y905, Y1015, and Y1062, and this was inhibited in a dose‑dependent manner by the TRK inhibitor (CEP‑701). Conversely, RET activation by ARTN in NBLS cells led to phosphorylation of TrkA. This suggests a physical association between RET and TRK proteins, and cross‑talk between these two receptor pathways. Finally, RET, GFR and TRK expression in primary tumors was investigated and a significant association between RET, its co‑receptors and TRK expression was demonstrated. Thus, the present data support a complex model of interacting neurotrophin receptor pathways in the regulation of cell growth and differentiation in NBs.

The multikinase inhibitor RXDX-105 is effective against neuroblastoma in vitro and in vivo.

Neuroblastoma is the most common extracranial solid tumor of childhood and accounts for 15% of all pediatric cancer-related deaths. New therapies are needed to improve outcomes for children with high-risk and relapsed tumors. Inhibitors of the RET kinase and the RAS-MAPK pathway have previously been shown to be effective against neuroblastoma, suggesting that combined inhibition may have increased efficacy. RXDX-105 is a small molecule inhibitor of multiple kinases, including the RET and BRAF kinases. We found that treatment of neuroblastoma cells with RXDX-105 resulted in a significant decrease in cell viability and proliferation in vitro and in tumor growth and tumor vascularity in vivo. Treatment with RXDX-105 inhibited RET phosphorylation and phosphorylation of the MEK and ERK kinases in neuroblastoma cells and xenograft tumors, and RXDX-105 treatment induced both apoptosis and cell cycle arrest. RXDX-105 also showed enhanced efficacy in combination with 13-cis-retinoic acid, which is currently a component of maintenance therapy for children with high-risk neuroblastoma. Our results demonstrate that RXDX-105 shows promise as a novel therapeutic agent for children with high-risk and relapsed neuroblastoma.

506P - TPX-0046 is a novel and potent RET/SRC inhibitor for RET-driven cancers

BackgroundOncogenic activation of the receptor tyrosine kinase (RTK) RET via point mutation or genomic rearrangement has been identified in multiple cancers, including medullary and papillary thyroid cancers, and non-small cell lung cancer (NSCLC). Two multi-kinase RET inhibitors vandetanib and cabozantinib have been approved for thyroid cancer. Current investigational RET inhibitors BLU-667 and LOXO-292 have demonstrated efficacy in RET aberrant NSCLC and thyroid cancers. Multiple resistance mutations have been reported from preclinical studies, including the gatekeeper mutation V804L to cabozantinib and solvent front mutations (SFMs) G810A/S to vandetanib. Given that current TKIs often lead to resistance, TPX-0046, a next generation RET inhibitor that is structurally differentiated and potent against various mutations, especially SFMs, was designed. MethodsA series of macrocyclic RET inhibitors were rationally designed and characterized in RET-related biological assays. TPX-0046 was further evaluated in RET-driven tumor models in vivo. ResultsTPX-0046 is a potent and selective next-generation RET/SRC inhibitor with a small and rigid macrocyclic structure that is structurally differentiated from current RET inhibitors. In enzymatic assays, TPX-0046 demonstrated low nanomolar potency against WT and many mutated RETs, as well as SRC, and is VEGFR2-sparing. TPX-0046 potently inhibited RET phosphorylation and cell proliferation in in-house engineered Ba/F3 KIF5B-RET, TT, and LC2/ad cells with IC50s of approximately 1nM. TPX-0046 is potent against the SFM G810R in Ba/F3 cell proliferation assay with a mean IC50 of 17nM, whereas comparable proxy molecules for BLU-667 and LOXO-292 have IC50s >500nM. TPX-0046 demonstrated marked anti-tumor efficacy in vivo in multiple RET-driven cancer cell-derived and patient-derived xenograft tumor models. ConclusionsTPX-0046 is a novel next generation RET/SRC inhibitor with favorable pharmaceutical properties and possesses potent in vitro and in vivo activity against diverse RET alterations, including the SFM G810R. The novel macrocyclic structure may provide opportunities to overcome treatment resistance from current RET inhibitors. TPX-0046 is currently in IND-enabling studies. Legal entity responsible for the studyTurning Point Therapeutics, Inc. FundingTurning Point Therapeutics, Inc. DisclosureE. Rogers: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. D. Zhai: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. W. Deng: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. X. Zhang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. D. Lee: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. J. Ung: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. J. Whitten: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. H. Zhang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. J. Liu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. T. Hu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. H. Zhuang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. Y. Lu: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. Z. Huang: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. A. Graber: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. Z. Zimmerman: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. R. Xin: Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics, Inc. J..J. Cui: Leadership role, Travel / Accommodation / Expenses, Shareholder / Stockholder / Stock options, Full / Part-time employment: Turning Point Therapeutics. All other authors have declared no conflicts of interest.

Enhancement of HGF-induced tubulogenesis by endothelial cell-derived GDNF.

Tubulogenesis, the organization of epithelial cells into tubular structures, is an essential step during renal organogenesis as well as during the regeneration process of renal tubules after injury. In the present study, endothelial cell-derived factors that modulate tubule formation were examined using an in vitro human tubulogenesis system. When human renal proximal tubular epithelial cells (RPTECs) were cultured in gels, tubular structures with lumens were induced in the presence of hepatocyte growth factor (HGF). Aquaporin 1 was localized in the apical membrane of these tubular structures, suggesting that these structures are morphologically equivalent to renal tubules in vivo. HGF-induced tubule formation was significantly enhanced when co-cultured with human umbilical vein endothelial cells (HUVECs) or in the presence of HUVEC-conditioned medium (HUVEC-CM). Co-culture with HUVECs did not induce tubular structures in the absence of HGF. A phospho-receptor tyrosine kinase array revealed that HUVEC-CM markedly enhanced phosphorylation of Ret, glial cell-derived neurotrophic factor (GDNF) receptor, in HGF-induced tubular structures compared to those without HUVEC-CM. HUVECs produced GDNF, and RPTECs expressed both Ret and GDNF family receptor alpha1 (co-receptor). HGF-induced tubule formation was significantly enhanced by addition of GDNF. Interestingly, not only HGF but also GDNF significantly induced phosphorylation of the HGF receptor, Met. These data indicate that endothelial cell-derived GDNF potentiates the tubulogenic properties of HGF and may play a critical role in the epithelial-endothelial crosstalk during renal tubulogenesis as well as tubular regeneration after injury.

Artemin and an Artemin-Derived Peptide, Artefin, Induce Neuronal Survival, and Differentiation Through Ret and NCAM.

Artemin (ARTN) is a neurotrophic factor from the GDNF family ligands (GFLs) that is involved in development of the nervous system and neuronal differentiation and survival. ARTN signals through a complex receptor system consisting of the RET receptor tyrosine kinase and a glycosylphosphatidylinositol-anchored co-receptor GFL receptor α, GFRα3. We found that ARTN binds directly to neural cell adhesion molecule (NCAM) and that ARTN-induced neuritogenesis requires NCAM expression and activation of NCAM-associated signaling partners, thus corroborating that NCAM is an alternative receptor for ARTN. We designed a small peptide, artefin, that could interact with GFRα3 and demonstrated that this peptide agonist induces RET phosphorylation and mimics the biological functions of ARTN – neuroprotection and neurite outgrowth. Moreover, artefin mimicked the binding of ARTN to NCAM and required NCAM expression and activation for its neurite elongation effect, thereby suggesting that artefin represents a binding site for NCAM within ARTN. We showed that biological effects of ARTN and artefin can be inhibited by abrogation of both NCAM and RET, suggesting a more complex signaling mechanism that previously thought. As NCAM plays a significant role in neurodevelopment, regeneration, and synaptic plasticity we suggest that ARTN and its mimetics are promising candidates for treatment of neurological disorders and warrant further investigations.

Reciprocal feedback regulation of ST3GAL1 and GFRA1 signaling in breast cancer cells

GFRA1 and RET are overexpressed in estrogen receptor (ER)-positive breast cancers. Binding of GDNF to GFRA1 triggers RET signaling leading to ER phosphorylation and estrogen-independent transcriptional activation of ER-dependent genes. Both GFRA1 and RET are membrane proteins which are N-glycosylated but no O-linked sialylation site on GFRA1 or RET has been reported. We found GFRA1 to be a substrate of ST3GAL1-mediated O-linked sialylation, which is crucial to GDNF-induced signaling in ER-positive breast cancer cells. Silencing ST3GAL1 in breast cancer cells reduced GDNF-induced phosphorylation of RET, AKT and ERα, as well as GDNF-mediated cell proliferation. Moreover, GDNF induced transcription of ST3GAL1, revealing a positive feedback loop regulating ST3GAL1 and GDNF/GFRA1/RET signaling in breast cancers. Finally, we demonstrated ST3GAL1 knockdown augments anti-cancer efficacy of inhibitors of RET and/or ER. Moreover, high expression of ST3GAL1 was associated with poor clinical outcome in patients with late stage breast cancer and high expression of both ST3GAL1 and GFRA1 adversely impacted outcome in those with high grade tumors.

GDNF mimetics delivery by porous silicon nanoparticles for improved Parkinson’s disease management

Parkinson’s disease is neurodegenerative movement disorder caused byprogressive degeneration of dopaminergic neurons in substantia nigra. Currently, there are no treatments to slow down, stop or reverse the loss of dopaminergic neuros. Glial cell line-derived neurotrophic factor (GDNF) is a protein that has been shown to prevent the death and help to repair the damaged neurons. However, GDNF is a large molecule (about 32 kDa) that cannot cross the blood brain barrier (BBB), and therefore needs to be delivered by intracranial administration, which requires complex and expensive stereotactic surgery. Generally, small molecules are easier to across the BBB than largemolecules. Thus, small molecules with similar biological activity to GDNF(named GDNF mimetics) might be a better approach to translate into the clinic.Several GDNF mimetics (molecular weight 500-600 g/mol), which are able to activate GDNF receptors and support the survival of dopaminergic neurons, have been found, but they have limited aqueous solubility. Porous silicon nanoparticles (PSi NPs) have the ability to improve the solubility and dissolution rate of poorly water-soluble drugs, as well as to enhance the drug permeation across biological barriers. In our study,GDNF mimetics were loaded into thermally oxidized-PSi (TOPSi) NPs, by which the solubility and dissolution rate of GDNF mimetics were considerably enhanced. Luciferase assay, GDNF receptor RET phosphorylation and internal signalling assay showed that the GDNF mimetics delivered by the TOPSi NPs exhibit higher activity than pristine GDNF mimetics, even in the absence of organic solvents.

Abstract 3018: RET rearrangements as promising therapeutic targets in breast cancer

Abstract Background: Receptor tyrosine kinase alterations have played a significant role in therapeutic decisions for cancer due to their oncogenic nature and response to targeted small molecule kinase inhibitors. Increased genomic profiling of tumors using hybrid-capture based next-generation sequencing approaches now reveal the presence of previously unknown fusions and alterations involving kinases in a diverse set of cancers. Here we report the presence and therapeutic significance of recurrent and novel fusions involving RET, a known oncogenic tyrosine kinase receptor, in breast cancer. Methods: Comprehensive genomic profiling on formalin-fixed, paraffin embedded patient tumor tissues was performed using FoundationOne platform that covers the entire coding region for 315 cancer-related genes and introns of 28 genes involved in rearrangements at a depth of 500-1000X (Foundation Medicine, MA). Out of 23 rearrangements, two representative RET fusion expression vectors were synthesized and expressed in non-tumorigenic cell lines (breast MCF10A and mouse 3T3 fibroblasts) and were evaluated for RET kinase signaling, drug response, and tumorigenicity. Results: RET gene fusions, the canonical NCOA4-RET and a novel, noncanonical RASGEF1A-RET fusion, were identified in two separate breast cancers and both include exons required to retain the intact kinase domain of Ret. The novel RASGEF1A-RET fusion includes the non-coding region of RASGEF1A potentially resulting in a truncated RET protein using an alternate internal start site in exon 11 of RET. In vitro characterization of both fusions expressed in mouse 3T3 and human MCF10a cell lines revealed constitutive kinase activation and subsequent downstream signaling as evidenced by phosphorylation of Ret, Erk and Akt. This is the first reported noncanonical RET rearrangement resulting in a 5’ truncated but functional RET kinase. Non-tumorigenic cell lines with stable expression of either rearrangement showed transformed phenotypes assessed by changes in morphology, enhanced growth rate, colony forming ability, and tumor formation in mice. RET fusion-transformed cells were exquisitely sensitive to treatment with RET inhibitors when evaluated in both short-term and long-term functional assays. NCOA4-RET was found by CGP in an index case of metastatic ER+/HER2+ breast cancer that had radiographic evidence of disease progression while on trastuzumab, pertuzumab, and anastrazole. Subsequent treatment with cabozantinib plus anastrazole led to a rapid clinical and radiographic response. Conclusion: CGP techniques involving hybrid-capture based approaches can identify previously unreported but recurrent RET gene fusions in breast cancer. Here, we show that RET fusions including both canonical and non-canonical complex rearrangements are functional and may represent promising therapeutic targets in selected breast cancer patients. Citation Format: Bhavna S. Paratala, Jeffrey S. Ross, Casey B. Williams, Whitney Petrosky, Kirstin A. Williams, Jon Chung, Sonia C. Dolfi, Shridar Ganesan, Siraj Ali, Brian Leyland-Jones, Kim M. Hirshfield. RET rearrangements as promising therapeutic targets in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3018. doi:10.1158/1538-7445.AM2017-3018

Role of ERBB signaling in RET-rearranged lung cancer and contribution of EGFR amplification to cabozantinib resistance.

11583 Background: Lung cancers driven by oncogenic RET fusions have lower response rates to targeted monotherapy such as cabozantinib (28%) relative to response rates typically observed in ALK- or ROS1- rearranged lung adenocarcinomas (60-80%). Methods: To identify targetable co-dependencies or cooperating pathways for RET fusion-positive lung cancers, we performed high-throughput chemical and genetic screens to find FDA-approved drugs or genes that when inhibited, would synergize with cabozantinib in RET fusion-positive lung cancer cell lines. In addition we performed NGS of a pair of pre-treatment and post-cabozantinib progression samples. Results: We identified EGFR siRNAs and anti-EGFR drugs as synergistic with cabozantinib. Combinations of drugs that target EGFR (cetuximab, afatinib, erlotinib, gefitinib, neratinib) and RET (cabozantinib, CEP-32496, lenvatinib, vandetanib) were more effective at reducing growth of RET cell lines than any single agent in vitro and in xenograft models. Cabozantinib treatment of RET fusion-positive cell lines inhibited EGFR and RET phosphorylation, an observation not seen in RET wild-type cell lines. Co-immunoprecipitation studies reveal that RET and EGFR interact. Ectopic expression of CCDC6-RET in NIH-3T3 or human bronchial epithelial cells resulted in upregulation of multiple ERBB receptors and ligands (not seen in a ROS1 fusion-positive cell line) and a concomitant increase in EGFR stability. Treatment with ERBB pathway ligands or overexpression of EGFR decreased sensitivity to cabozantinib in two RET fusion-positive cell lines. Finally, sequencing of a pair of pre-treatment and post-progression samples from a lung cancer patient treated with cabozantinib revealed acquired amplification of EGFR in the latter sample. Conclusions: Taken together, these results suggest that the tumorigenic potential of RET fusion oncogenes is dependent on deregulation of ERBB-activated pathways and that a combination of RET and EGFR drugs could be more effective in treating RET fusion-positive tumors. Moreover, amplification of EGFR is a potential driver of resistance to cabozantinib in RET-rearranged lung cancers.

Synthesis and structure–activity relationship study of pyrazolo[3,4-d]pyrimidines as tyrosine kinase RET inhibitors

Three series of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and evaluated as RET kinase inhibitors. Compounds 23a and 23c were identified to show significant activity both in the biochemical and the BaF3/CCDC6-RET cell assays. Compound 23c was found to significantly inhibit RET phosphorylation and down-stream signaling in BaF3/CCDC6-RET cells, confirming its potent cellular RET-targeting profile. Different from other RET inhibitors with equal potency against KDR that associated with severe toxicity, 23c did not show significant KDR-inhibition even at the concentration of 1μM. These results demonstrated that 23c is a potent and selective RET inhibitor.