Smoothened Inhibitor Research Articles

Discovery of N-substituted-2-oxoindolin benzoylhydrazines as c-MET/SMO modulators in EGFRi-resistant non-small cell lung cancer.

Non-small cell lung cancer (NSCLC), the leading cause of cancer-related mortality worldwide, poses a formidable challenge due to its heterogeneity and the emergence of resistance to targeted therapies. While initially effective, first- and third-generation EGFR-tyrosine kinase inhibitors (TKIs) often fail to control disease progression, leaving patients with limited treatment options. To address this unmet medical need, we explored the therapeutic potential of multitargeting agents that simultaneously inhibit two key signalling pathways, the mesenchymal-epithelial transition factor (c-MET) and the G protein-coupled receptor Smoothened (SMO), frequently dysregulated in NSCLC. By employing a combination of in silico drug repurposing and structure-based structure-activity relationship (SAR) studies, we identified and developed novel c-MET/SMO-targeting agents with antiproliferative activity against first- as well as third-generation EGFR-TKI-resistant NSCLC cells suggesting a synergistic effect arising from the simultaneous inhibition of c-MET and SMO.

Treatment with GT1708F, an Oral Smoothened Inhibitor, in Patients with Myeloid Malignancies: A Phase I Study

Introduction: Activation of the Hedgehog signaling pathway has been correlated with the development and disease progression of myeloid leukemia. This Phase I clinical trial aimed to evaluate the dose-limiting toxicities (DLT), maximum tolerated dose (MTD), safety and tolerability of GT1708F in patients with myeloid malignancies. Methods: This Phase I study enrolled patients with myeloid malignancies who were refractory, resistant or intolerant to previous treatments at three centers in China. Eligible patients were enrolled at each dose cohort to evaluate DLTs during the first continuous 28-day dosing period. The dose levels were determined employing the escalation with overdose control (EWOC) method, guided by an adaptive Bayesian logical analysis model (ABLRM). The decision to escalate the dosage to the subsequent cohort was informed by the recommendations of the safety monitoring committee after each round. Pharmacokinetic blood samples were collected in the first cycle of the treatment. Results: As of May 25, 2023, 18 patients have been enrolled in 7 dose cohorts: 15 with acute myeloid leukemia (AML) and 3 with myelodysplastic syndromes (MDS). Patients received GT1708F once daily orally at 20 mg QD (n=1), 40 mg QD (n=1), 80 mg QD (n=4), 120 mg QD (n=3), 180 mg QD (n=3), 240 mg QD (n=3), 320 mg QD (n=3). No DLT was reported across these dose cohorts and the MTD was not reached. 17(93.3%) patients experienced at least one treatment-emergent adverse event (TEAE). 13 (72.2%) patients experienced at least one treatment related AE (TRAE). 11(61%) patients experienced at least one TEAE of Grade 3 or higher in severity, 4 (22.2%) of whom had TRAE(s) including lung infection, anemia, pharyngitis, and creatine kinase increased. Eight patients experienced 13 serious adverse events (SAE), two of which were assessed by the investigator as possibly related to GT1708F, both being lung infections. 15 patients completed at least one efficacy evaluation. No patient achieved complete or partial remission while 5 AML patients achieved stable disease with one patient lasting for 6 cycles and 2 patients lasting for 3 cycles. GT1708F is rapidly absorbed in the body with a peak time of 0.5-1hr after a single oral dose. As the dose increased from 20mg QD to 320 QD, the maximum concentration of the drug in the body increased linearly, showing quasi-linear pharmacokinetic characteristics. Conclusions: Our study indicated good safety and tolerability of GT1708F in patients with myeloid malignancies. In addition, a potential efficacy signal of GT1708F for these patients was observed, providing a rationale for further exploration in combination with other therapeutic modalities.

CSIG-03. SHH PATHWAY ACTIVATION IN DEDIFFERENTIATION DURING TUMOUR PROGRESSION IN MPNST

Abstract BACKGROUND Malignant peripheral nerve sheath tumours (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previous work conducted in our lab used unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumours to uncover two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1 pathway activation). Further, single nuclear RNA-sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. PURPOSE & HYPOTHESIS To examine the expression of transcription factors (TWIST1, SOX9, SNAI2, OTX2, PAX3, and PAX6) known to play canonical roles in the early neural crest cell specification in MPNST-G1 cells. We speculate that MPNST-G1 cells will display overexpression of these transcription factors compared to MPNST-G2 cells and that Sonidegib (SMO inhibitor) will revert dedifferentiation by decreasing activation of the SHH pathway. METHODS Dedifferentiation transcription factor expression and the effects of SMO activation and inhibition in MPNST-G1 and MPNST-G2 cells were analyzed using RT-PCR and western blotting. Alamar blue and Trypan blue assays were used to determine the effect of SMO inhibition on proliferation. RESULTS Compared to MPNST-G2 cells, MPNST-G1 cells displayed elevated expression of dedifferentiation transcription factors, SMO inhibition was able to reverse these effects. Conversely SMO activation induced the expression of these factors and induced an increase in proliferation. CONCLUSIONS The SHH pathway activation promotes the expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options for these lethal cancers.

RBIO-04. INHIBITING THE SHH PATHWAY AND PARP1 IN COMBINATION ENHANCES THE RADIATION SENSITIVITY OF PEDIATRIC SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract Medulloblastoma accounts for 20-25% of all pediatric brain tumors. These patients undergo craniospinal and focal radiation therapy during their treatment, resulting in negative long-term consequences such as bone and tissue hypoplasia and decreased neurocognitive functioning. One of the most concerning effects is the significantly increased risk for secondary radiation induced cancers later in life. Thus, there is a critical need for novel approaches to radiation therapy that can effectively target and kill tumor cells while minimizing long lasting toxic effects. Here, we investigate a novel approach to radiation therapy that relies on the combination of two classes of therapeutic agents, Sonic Hedgehog (SHH) inhibitors and Poly (ADP-ribose) Polymerase (PARP) inhibitors. The SHH pathway plays a key role in medulloblastoma tumorigenesis and affects the radiation sensitivity of various cancer types such as basal cell carcinoma. The PARP family, specifically PARP1, plays a key role in DNA damage repair and affects the radiation sensitivity of pediatric brain cancers such as high-grade glioma and medulloblastoma. We hypothesize that combining these approaches by inhibiting Smo and Gli1 in the SHH pathway, and PARP1 simultaneously, will enhance medulloblastoma tumor cell killing following radiation therapy. We found that in vitro treatment with Sonidegib, GANT61, and Veliparib alone and in combination with was sufficient to significantly decrease tumor cell viability and self-renewal as measured by clonogenic survival assays (p < 0.01). 24 hours following these targeted treatments, 2 Gy of radiation was administered which resulted in a greater degree of decreased cell viability and self-renewal (p < 0.01), as well as in increased cell death. Together, this data suggests that inhibition of Smo, Gli1, and PARP1 either alone as single agents, or in combination is a promising therapeutic strategy for increasing the radiation sensitivity of pediatric SHH medulloblastoma.

CSIG-02. SHH PATHWAY ACTIVATION DRIVES NEURAL CREST CELL SIGNATURE IN A SUBGROUP OF MPNSTS

Abstract INTRODUCTION Neurofibromatosis Type 1 is hereditary tumor predisposition syndrome that results in the development of innumerable nerve sheath tumors that fall within the spectrum of benign, premalignant and malignant lesions. Our previous work has demonstrated that SHH pathway activation drives malignant transformation in a subset of malignant peripheral nerve sheath tumors (MPNSTs). Here, we demonstrate that SHH pathway activation induces a neural crest cell-like state in a subset of MPNSTs. METHODS We performed single nuclear RNA sequencing on 6 tumors (5 MPNSTS and 1 atypical neurofibroma). We performed in vitro experiments to assess the correlation between SHH pathway activation and neural crest signatures. We inhibited SHH pathway with sonidegib, a SMO inhibitor, in MPNST cell lines and performed RT-PCR to assess markers of neural crest cell signatures. In addition, we assessed cellular viability with treatment. RESULTS We observed that cells that express SMO at high levels, with SHH pathway activation, express neural crest cell makers. Single cell trajectory analysis further demonstrates a pseudotemporal continuum from atypical neurofibromas (Schwann cells), MPNSTs (Schwann cell precursor cells) and other MPNSTs (neural crest cells), which supports that these tumors fall along the developmental trajectory of neural crest cell lineage. SHH pathway activity was high in S462TY MPNST cell line and low in STS-26T MPNST cell line. S462TY also demonstrated prominent elevation in expression of neural crest cell markers TWIST1, SOX9, SNAI2, OTX2, PAX4 and PAX6 . We found that treatment with sonidegib treatment attenuates the expression of neural crest cell signatures. Finally, inhibiting SHH pathway activation reduced cellular viability in MPNST cell lines. CONCLUSION SHH pathway activation drives dedifferentiation into a neural crest cell-like state in a subset of MPNSTs. We confirm that inhibiting SHH pathway activity in a subset of MPNSTS prevent growth and malignant progression, providing a rational for future clinical trials.

Deciphering the Role of RAS Pathway Mutations in the Biology of Human Acute Myeloid Leukemia Using In Vivo Models

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy for which the mutational profile of the leukemic cells is the most important prognostic factor for disease-free survival and overall survival. The failure of chemotherapy to completely eradicate the leukemic clones is the most common cause of death in AML. The genetic events that can contribute to chemoresistance are therefore important factors for the clinical outcome, but the relative importance of these events on chemoresistance and persistence of leukemia stem cells (LSC) remains poorly defined. MLL rearranged AMLs account for 5-10% of all AMLs and the RAS pathway is the most frequently mutated pathway in this sub-group of AMLs. The goal of this project is to study the role and biology of RAS pathway mutations in clonal evolution in human MLL-rearranged AML. Methods We have previously shown that human cord blood CD34+ cells transduced with a retrovirus encoding the MLL-AF9 fusion gene with an EGFP maker generate human AML when injected into immunodeficient NSG-SGM3 mice. Using a similar experimental design, we added a second hit to the CD34 transduced cells before their injection into NSG-SGM3 mice. MLL-AF9 cells were electroporated with Cas9 enzyme for the control and with Cas9 enzyme plus a single guide RNA (sgRNA) designed to induce the KRAS G13D or the NRAS G12D mutation. Mice were sacrificed when they showed sign of disease and the bone marrow and spleen were collected and analyzed. Kaplan-Meier curves, flow cytometry analyses, RNA and exome sequencing and secondary transplantation were performed on the samples. Results Mice injected with MLL-AF9 + KRAS G13D got sick around day 80 after injection and around day 60 for MLL-AF9 NRAS G12D, whereas it took ~150 days for control mice (MLL-AF9 KRAS WT) to get sick (fig 1). In multiple experiment, the latency was roughly cut by half by the KRAS G13D and NRAS G12D mutations. The cell surface phenotype and morphology were not significantly different between MLL-AF9 WT and MLL-AF9 KRAS/NRAS mutated. The variant allele frequency (VAF) for the KRAS mutation invariably increased from the day of injection to the day of sacrifice, where the percentages varied between 20-40%, which is typically observed in AML patients. In contrast, the VAF for NRAS mutated cells was lower (3-9%) but seems to be very aggressive into mice. We performed limiting dilution analyses in secondary mice to quantify the LSC frequencies in MLL-AF9 WT and KRAS G13D / NRAS G12D leukemia. The average frequencies of control MLL-AF9 AMLs were between 1/510 000 to 1/1 450 000, while MLL-AF9+KRAS G13D AMLs frequencies were >1/45 000 and 1/7 800 for MLL-AF9+NRAS G12D AMLs, demonstrating that these 2 mutations increase the LSC frequency by >20-100 fold in our modelled MLL-AF9 AML. Using transcriptome analyses, we looked at the gene expression changes induced by the KRAS G13D mutation in comparison to MLL-AF9 leukemias. These experiments are in progress for the NRAS G13D mutation. Many more genes were overexpressed in MLL-AF9 KRAS G13D vs MLL-AF9 KRAS WT than down regulated. Genes from the hedgehog (Hh) pathway such as Patched1 (PTCH1) and Smoothened (SMO) were amongst the genes the most differentially overexpressed by the KRAS mutation. It is particularly interesting because they have been shown important in stem cell biology and could explain the increase in LSC. Some SMO inhibitors have been tested in clinical trial and Glasdegib is FDA approved in combination with low dose cytarabine in elderly patients. Its effect on MLL leukemias or on KRAS mutated AMLs has not been tested/reported in a clinical study. Experiments are currently in progress to test the effect of Glasgebib on engraftment of MLL-AF9 KRAS mutated cells in our experimental setting. Single cell RNA sequencing data on the three conditions will be presented at the meeting. Given the success of generating these two point mutations, we are currently generating more RAS pathway mutations, including other KRAS mutations, PNTP11 and NF1 mutations. Conclusion These experiments showed: 1) It is possible to induce 2 oncogenic hits in human primary cells and get leukemia in vivo; 2) the KRAS G13D and NRAS G12D mutations shorten the latency of the disease and 3) increase the LSC frequency in secondary mice; 4) a possible involvement of the Hh pathway on stemness/LSC in RAS mutated cells; 5) our experimental approach is robust and very promising to decipher the RAS pathway in human MLL leukemias.

Integrated PK/PD Modeling Relates Smoothened Inhibitor Biomarkers to The Heterogeneous Intratumor Disposition of Cetuximab in Pancreatic Cancer Tumor Models

Therapeutic antibodies have shown little efficacy in the treatment of pancreatic ductal adenocarcinomas (PDAC). Tumor desmoplasia, hypovascularity, and poor perfusion result in insufficient tumor cell exposure, contributing to treatment failure. Smoothened inhibitors of hedgehog signaling (sHHi) increase PDAC tumor permeability, perfusion, and drug delivery, and provide a tool to develop a quantitative, mechanistic understanding as to how the temporal dynamics of tumor priming can impact intratumor distribution of monoclonal antibodies (mAb). A linked pharmacokinetic (PK)/pharmacodynamic (PD) model was developed to integrate the plasma and tumor PK of a sHHi priming agent with its effects upon downstream stromal biomarkers Gli1, hyaluronic acid, and interstitial fluid pressure in PDAC patient-derived xenograft (PDX) tumors. In parallel, in situ tumor concentrations of cetuximab (CTX: anti-epidermal growth factor receptor; EGFR) were quantified as a marker for tumor delivery of mAb or antibody-drug conjugates. A minimal, physiologically-based pharmacokinetic (mPBPK) model was constructed to link sHHi effects upon mechanistic effectors of tumor barrier compromise with the intratumor distribution of CTX, and CTX occupancy of EGFR in tumors. Integration of the mPBPK model of mAb deposition and intratumor distribution with the PK/PD model of tumor responses to priming not only identified physiological parameters that are critical for tumor antibody distribution, but also provides insight into dosing regimens that could achieve maximal tumor disposition of therapeutic antibodies under conditions of transient PDAC tumor permeability barrier compromise that mechanistically-diverse tumor priming strategies may achieve.

Increasing Ciliary ARL13B Expression Drives Active and Inhibitor-Resistant Smoothened and GLI into Glioma Primary Cilia.

ADP-ribosylation factor-like protein 13B (ARL13B), a regulatory GTPase and guanine exchange factor (GEF), enriches in primary cilia and promotes tumorigenesis in part by regulating Smoothened (SMO), GLI, and Sonic Hedgehog (SHH) signaling. Gliomas with increased ARL13B, SMO, and GLI2 expression are more aggressive, but the relationship to cilia is unclear. Previous studies have showed that increasing ARL13B in glioblastoma cells promoted ciliary SMO accumulation, independent of exogenous SHH addition. Here, we show that SMO accumulation is due to increased ciliary, but not extraciliary, ARL13B. Increasing ARL13B expression promotes the accumulation of both activated SMO and GLI2 in glioma cilia. ARL13B-driven increases in ciliary SMO and GLI2 are resistant to SMO inhibitors, GDC-0449, and cyclopamine. Surprisingly, ARL13B-induced changes in ciliary SMO/GLI2 did not correlate with canonical changes in downstream SHH pathway genes. However, glioma cell lines whose cilia overexpress WT but not guanine exchange factor-deficient ARL13B, display reduced INPP5e, a ciliary membrane component whose depletion may favor SMO/GLI2 enrichment. Glioma cells overexpressing ARL13B also display reduced ciliary intraflagellar transport 88 (IFT88), suggesting that altered retrograde transport could further promote SMO/GLI accumulation. Collectively, our data suggest that factors increasing ARL13B expression in glioma cells may promote both changes in ciliary membrane characteristics and IFT proteins, leading to the accumulation of drug-resistant SMO and GLI. The downstream targets and consequences of these ciliary changes require further investigation.

P20.16.B FIRST-LINE TREATMENT OF MEDULLOBLASTOMA IN ADOLESCENTS AND ADULTS - UPDATE ON THE EORTC 1634-BTG/NOA-23 PHASE II TRIAL

Abstract BACKGROUND Medulloblastoma is a common brain malignancy in children. Patients after puberty are rare and bear an intermediate prognosis. Standard treatment consists of maximal resection plus radio-chemotherapy. Treatment toxicity is high and produces disabling long-term side effects. The sonic hedgehog (SHH) subgroup is highly overrepresented in the post-pubertal and adult population and can be targeted by smoothened (SMO) inhibitors. No practice-changing prospective randomized data have been generated in adults so far. MATERIAL AND METHODS EORTC 1634-BTG/NOA-23 trial randomizes patients between standard-dose vs. reduced-dosed craniospinal radiotherapy and SHH-subgroup patients between the SMO inhibitor sonidegib (OdomzoTM, Sun Pharmaceuticals Industries, Inc., New York, USA) and standard chemotherapy. Based on data that suggest equal efficacy, a reduction of toxicity with dose-reduced CSI while maintaining efficacy is investigated by using 35.2 Gy in the standard vs. 24.6 Gy in the experimental arm. Chemotherapy consists of vincristine during radiotherapy, followed by a maximum of six cycles of lomustine, cisplatin and vincristine of six-weekly cycles. Sonidegib is added for patients with SHH-activated medulloblastoma. An increase in efficacy in the SHH subgroup is the primary efficacy endpoint. A number of translational research projects are performed alongside the trial. RESULTS The trial aims at developing a personalized, genotype-based, intensity-modulated therapy for post-pubertal and adult patients with newly diagnosed medulloblastoma. We are investigating tumor tissue, blood, and cerebrospinal fluid as well as magnetic resonance imaging and radiotherapy plans to generate information that helps to further improve treatment outcomes. Long-term follow-up is performed to monitor classic side effects of therapy, but also health-related quality of life, cognition, social and professional outcome, and reproduction and fertility. The 1634-BTG/NOA-23 trial started recruitment in November 2022. Updated information on recruitment as well as on demographic data of recruited patients will be presented at the EANO annual meeting. CONCLUSION EORTC 1634-BTG/NOA-23 is the first trial worldwide that prospectively enrolls post-pubertal and adult patients with medulloblastoma according to their molecular subgroup in a randomized design. Translational subprojects will enable us to evaluate events on several layers to gain deeper insights into subgrouping, risk stratification, therapeutic targets, resistance mechanisms, and the toxicity of treatments. The trial will therefore generate unprecedented data that will be translated into treatment changes in post-pubertal patients with medulloblastoma and will help to design future clinical trials.

P04.04.A GENOME-WIDE CRISPR/CAS9 KNOCKOUT SCREENS REVEAL EPIGENETIC REGULATORS AS DRUGGABLE TARGETS FOR SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract BACKGROUND Sonic hedgehog medulloblastoma (SHH-MB) is a malignant pediatric brain tumor that accounts for 30% of all MBs and is characterized by constitutive activation of the SHH signaling pathway. Besides the standard-of-care therapies, current therapies are targeting Smoothened (SMO) receptor, in order to inhibit the pathway. However, acquired or de novo resistance against SMO inhibition limits its efficacy. Therefore, our aim is to unravel novel therapeutic targets for SHH-MB, potentially beyond the SHH pathway. MATERIAL AND METHODS We performed genome-wide CRISPR/Cas9 knockout screens, in order to decipher cancer dependencies and synthetic lethal interactors for SHH-MB. The hits identified were first validated genetically and pharmacologically in in vitro assays, while further molecular analyses including RNAseq and differential methylation were also conducted. Moreover, the identified hits were evaluated in in vivo mouse models representing normal cerebellar, as well as SHH-MB development. RESULTS Functional genomics identified members of the epigenetic machinery, such as Dnmt1 and Smarca5 as context-specific dependencies for SHH-MB. We prove that DNMT1 inhibition is efficacious against SMO-resistant and sensitive SHH-MB cell lines, by inhibiting SHH pathway output, as shown via RNAseq. A further knockout drug screen unraveled novel synthetic lethal interactors for DNMT1 inhibitors. Genetic ablation and pharmacological inhibition of epigenetic regulators prolongs survival of established mouse models of SHH-MB. CONCLUSION Summarizing, our data indicate inhibitors of epigenetic regulators as novel therapeutic targets for SHH-MB irrespective of mutational drivers within the SHH pathway. We further propose combinatorial treatment approaches for this tumor entity that involve DNMT1 inhibitors.

Hedgehog signaling is essential in the regulation of limb regeneration in the Chinese mitten crab, Eriocheir sinensis.

Tissue autotomy is a unique adaptive response to environmental stress, followed by regeneration process compensating for the loss of body parts. The crustaceans present remarkable activity of appendage autotomy and regeneration, however, the molecular mechanism is still unclear. In this study, the Eriocheir sinensis Hedgehog (EsHH) and Smoothened (EsSMO) were identified in the regenerative limbs, and the function of Hedgehog signaling pathway on limb regeneration was evaluated. At the blastema growth stage of limb regeneration, the expression of EsHH and EsSMO was up-regulated in response to limb autotomy stress, and down-regulated at blastema differentiation stage. To clarify the effect of Hedgehog pathway during limb regeneration, the regenerative efficiency was evaluated with Smoothened inhibitor cyclopamine or RNAi (ds-HH) injection. We observed that the regenerative efficiency was significantly repressed with blockage of Hedgehog pathway at both the basal growth stage and the proecdysial growth stage, which was indicated by the delay of wound healing and blastema growth, as well as a decrease in the size of newly formed limbs. In addition, gene expression and BrdU incorporation assay showed that the proliferation and myogenic differentiation of blastema cells were suppressed with either cyclopamine or ds-HH injection. Thus, these results suggest that Hedgehog signaling pathway is essential for the establishment of limb regeneration in E. sinensis through promoting the proliferation and myogenic differentiation of blastema cells.

Smoothened mediates medaka spermatogonia proliferation via Gli1-Rgcc-Cdk1 axis†.

The proliferation of spermatogonia directly affects spermatogenesis and male fertility, but its underlying molecular mechanisms are poorly understood. In this study, Smoothened (Smo), the central transducer of Hedgehog signaling pathway, was characterized in medaka (Oryzias latipes), and its role and underlying mechanisms in the proliferation of spermatogonia were investigated. Smo was highly expressed in spermatogonia. In ex vivo testicular organ culture and a spermatogonial cell line (SG3) derived from medaka mature testis, Smo activation promoted spermatogonia proliferation, while its inhibition induced apoptosis. The expression of glioma-associated oncogene homolog 1 (gli1) and regulator of cell cycle (rgcc) was significantly upregulated in SG3 after Smo activation. Furthermore, Gli1 transcriptionally upregulated the expression of rgcc, and Rgcc overexpression rescued cell apoptosis caused by Smo or Gli1 inhibition. Co-immunoprecipitation assay indicated that Rgcc could interact with cyclin-dependent kinase 1 (Cdk1) to regulate the cell cycle of spermatogonia. Collectively, our study firstly reveals that Smo mediates the proliferation of spermatogonia through Gli1-Rgcc-Cdk1 axis. In addition, Smo and Gli1 are necessary of the survival of spermatogonia. This study deepens our understanding of spermatogonia proliferation and survival at the molecular level, and provides insights into male fertility control and reproductive disease treatment.

Development of a Patient-Reported Outcome Measure (PROM) for Dysgeusia During Treatment With Smoothened (SMO) Inhibitors for Basal Cell Carcinomas: The SMO-iD Questionnaire.

Dysgeusia may occur during conventional or target-therapies and affect patients adherence-to-treatment. Therefore, it should be monitored to improve clinical outcome. To date, available questionnaires on dysgeusia relate to traditional antineoplastics and do not apply to target-therapies as the pathogenetic mechanism and clinical expression differ. To develop a patient-reported outcome measure (PROM) to screen for and monitor the occurrence and severity of dysgeusia in patients under Smoothened (SMO) inhibitors: the SMO-iD questionnaire. Patients with locally advanced basal cell carcinomas referring dysgeusia under SMO inhibitors at the University Hospital of Naples Federico II, were enrolled between January-December 2020. The PROM was elaborated based on chemotherapy-induced dysgeusia (CiTas) scale (development phase) and then validated by measuring internal consistency and reliability (validation phase). Thirty-nine patients were enrolled and interviewed every 8 weeks. In the first phase, 160 CiTas questionnaires were collected, and the SMO-iD questionnaire was developed. In the second phase, 195 SMO-iD questionnaires were recorded, and reliability and validity assessed. Cronbach alpha was 0.89. The SMO-iD questionnaire is a validated questionnaire that shows high face and content validity as well as high internal consistency and reliability. Hence, it may be introduced in daily clinical setting to monitor dysgeusia in patients under SMO-inhibitors.

SHH PATHWAY ACTIVATION IN DEDIFFERENTIATION DURING TUMOR PROGRESSION IN MPNST

Abstract Malignant peripheral nerve sheath tumours (MPNST) are highly aggressive sarcomas with little progress on outcomes and treatment strategies. Previous work conducted in our lab used unsupervised analyses of methylome and transcriptome profiles of 108 peripheral nerve sheath tumours to uncover two subgroups of MPNSTs that predict progression-free survival, MPNST-G1 (characterized by SHH pathway activation) and MPNST-G2 (characterized by WNT/ß-catenin/CCND1 pathway activation). Further, single nuclear RNA-sequencing revealed that MPNST-G1 and MPNST-G2 cells resemble neural crest-like and Schwann cell precursor-like cells, respectively. Purpose & Hypothesis: To examine the expression of transcription factors (TWIST1, SOX9, SNAI2, OTX2, PAX3, and PAX6) known to play canonical roles in the early neural crest cell specification in MPNST-G1 cells. We speculate that MPNST-G1 cells will display overexpression of these transcription factors compared to MPNST-G2 cells and that Sonidegib (SMO inhibitor) will revert dedifferentiation by decreasing activation of the SHH pathway. METHODS: Dedifferentiation transcription factor expression and the effects of SMO activation and inhibition in MPNST-G1 and MPNST-G2 cells were analyzed using RT-PCR and western blotting. Alamar blue and Trypan blue assays were used to determine the effect of SMO inhibition on proliferation. RESULTS: Compared to MPNST-G2 cells, MPNST-G1 cells displayed elevated expression of dedifferentiation transcription factors, SMO inhibition was able to reverse these effects. Conversely SMO activation induced the expression of these factors and induced an increase in proliferation. CONCLUSIONS: The SHH pathway activation promotes the expression of important transcription factors in dedifferentiation. This finding provides insights into the transformation process of MPNST and novel therapeutic options for these lethal cancers.

MDB-36. SONIDEGIB, GANT61, AND VELIPARIB TREATMENT BOTH ALONE AND IN COMBINATION INCREASES RADIATION SENSITIVITY IN PEDIATRIC SONIC HEDGEHOG MEDULLOBLASTOMA

Abstract Medulloblastoma is the most common malignant brain tumor in children, accounting for 20-25% of all pediatric brain tumors. The current standard of care includes both craniospinal and focal radiation therapy which lead to negative long term consequences in the pediatric population such as bone and tissue hypoplasia, decreased neurocognitive functioning, as well as an increased risk for secondary radiation induced cancers. Thus, there is a need for novel approaches to radiation therapy that can effectively target and kill tumor cells while minimizing radiation toxicity in healthy tissue. Here, we investigate a new approach to radiation therapy relying on two classes of therapeutic agents, Sonic Hedgehog (SHH) inhibitors and Poly (ADP-ribose) Polymerase (PARP) inhibitors. The dysregulation and constitutive activation of the SHH signaling pathway accounts for ~30% of all medulloblastomas. In addition to playing a role in tumorigenesis, the SHH pathway is known to play a role in the radiation sensitivity of various cancer types. PARP1 is known to play a key role in DNA damage repair, and it has previously been shown to affect the radiation sensitivity of pediatric brain cancers including high grade glioma, medulloblastoma, and ependymoma. We hypothesize that both inhibiting the SHH pathway at Smoothened (Smo) or Gli1 and inhibiting PARP1 will enhance medulloblastoma tumor cell killing following radiation therapy. We found that in vitro treatment with Sonidegib, GANT61, and Veliparib alone without radiation was sufficient to decrease tumor cell viability and self-renewal. When followed by radiation, pre-treatment with Sonidegib, GANT61, and Veliparib, both alone and in combination, resulted in further decreased tumor cell viability and self-renewal. These data suggest that inhibition of Smo, Gli1, or PARP1 alone or in combination is a promising therapeutic strategy for increasing the radiation sensitivity of pediatric SHH medulloblastoma.

Mechanisms of hedgehog, calcium and retinoic acid signalling pathway inhibitors: Plausible modes of action along the MLL–EZH2-p53 axis in cellular growth control

Understanding the molecular mechanism(s) of small compounds in cellular growth control are essential for using those against the disease(s). Oral cancers exhibit a very high mortality rate due to higher metastatic potential. Aberrant EGFR, RAR, HH signalling, enhanced [Ca2+] and oxidative stress are some of the important characteristics of oral cancer. So, we target these for our study. Herein, we tested the effect of fendiline hydrochloride (FH) as an LTCC Ca2+-channel inhibitor, erismodegib (a SMO inhibitor of HH-signalling) and all-trans retinoic acid (RA) inducer of RAR signalling that causes cellular differentiation. OCT4 activating compound (OAC1) counters differentiation and induces stemness properties. Cytosine β-D arabinofuranoside (Cyto-BDA), a DNA replication inhibitor was used to reduce high proliferative capacity. Treatment of FaDu cells with OAC1, Cyto-BDA and FH increase G0/G1 population by 3%, 20% and 7% respectively, and lead to reduction of cyclin D1, CDK4/6 levels. Erismodegib arrests the cells in S-phase with reduced cyclin-E1&A1 levels, whereas RA-treatment causes G2/M phase arrest with reduced cyclin-B1. There was a decrease in the expression of EGFR and mesenchymal markers, Snail/Slug/Vim/Zeb/Twist, and increased E-cadherin expression in all the drug treatments, indicating a reduction in proliferative signal and EMT. Enhanced MLL2 (Mll4) and reduced EZH2 expression associated overexpression of p53 and p21 were traced out. We conclude that these drugs impact expression of epigenetic modifiers by modulating signalling pathways and the epigenetic modifiers then controls the expression of cell cycle control genes, including p53 and p21.

Ligand-based 3D pharmacophore modeling, virtual screening, and molecular dynamic simulation of potential smoothened inhibitors.

The Hedgehog (Hh) signaling pathway is a crucial regulator of various cellular processes. Dysregulated activation of the Smoothened (SMO) oncoprotein, a key component of the Hh pathway, has been implicated in several types of cancer. Although SMO inhibitors are important anti-cancer therapeutics, drug-resistant SMO mutants have emerged, limiting their efficacy. This study aimed to discover stable SMO inhibitors for both wild-type and mutant SMOs, using a 12-feature pharmacophore model validated for virtual screening. One lead compound, LCT10312, was identified with high affinity to SMO and showed a significant conformational change in the SMO structure upon binding. Molecular dynamic simulation revealed stable interaction of LCT10312 with SMO and large atom motions, indicating SMO structural fluctuation. The lead compound showed high predicted binding scores to several clinically relevant SMO mutants. A ligand-based pharmacophore model was developed from 25 structurally clustered SMO inhibitors using LigandScout v3.12 software and virtually screened for hit identification from a library of 511,878 chemicals. Molecular docking was employed to identify potential leads based on SMO affinities. Molecular dynamic simulation (MDS) with GROMACS v5.1.4 was performed to analyze the structural changes of SMO oncoprotein upon binding lead compound(s) and cyclopamine as the control for 100 ns. The binding affinity of lead compound(s) was predicted on clinical and laboratory SMO mutants.

Abstract 3569: An animal model of GLI2-amplified medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant brain tumor affecting children. Among the known subtypes of MB, GLI2-amplified SHH-MB associated with P53 mutations has the worst prognosis, and a 5-year survival rate of less than 30%. Moreover, GLI2-amplified MBs are non-responsive to SMO inhibitors, the only targeted treatment option available for SHH-MB. To address this treatment gap, there is an urgent need to identify novel potential therapeutic targets, an effort that requires better understanding of key cellular and molecular mechanisms underlying GLI2-amplified MB. To that end, we generated a mouse model of GLI2-amplified MB and identified GL12 as the critical driver of tumorigenesis, and granule cell progenitors (GCPs) as the cells of tumor origin. We further found that GLI2 specifically drives embryonic, but not neonatal, Math1+ GCPs to form SHH-MB tumors. An scRNA-seq analysis revealed MAPK pathway activation specifically in embryonic Math1+ GCPs. The MAPK pathway was activated in mouse and human GLI2-driven MB tumors, and a MEK/ERK inhibitor significantly inhibited GLI2-driven MB in vivo. Based on these findings, we hypothesize that GLI2-driven MB originates from a specific population of Math1+ GCPs during a discrete temporal interval in cerebellar development, and that targeting MAPK/MEK/ERK pathway components represents a novel approach to treatment of GLI2-amplified MB. Citation Format: Yanxin Pei. An animal model of GLI2-amplified medulloblastoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3569.

B13, a well-tolerated inhibitor of hedgehog pathway, exhibited potent anti-tumor effects against colorectal carcinoma in vitro and in vivo

Abnormal activation of Hedgehog (Hh) signaling pathway mediates the genesis and progression of various tumors [1]. Currently, three drugs targeting the Hh signaling component Smoothened (Smo) have been marketed for the clinical treatment of basal cell tumors or acute myeloid leukemia. However, drug resistance is a common problem in those drugs, so the study of Smo inhibitors that can overcome drug resistance has important guiding significance for clinical adjuvant drugs. MTT assay, clone formation assay and EdU assay were used to detect the proliferation inhibitory activity of the drugs on tumor cells. The effect of B13 on cell cycle and apoptosis were detected by flow cytometry. An acute toxicity test was used to detect the toxicity of B13 in vivo, and xenograft tumor model was used to detect the efficacy of B13 in vivo. The binding of B13 to Smo was studied by BODIPY-cyclopamine competitive binding assay and molecular docking. The effect of B13 on the expression and localization of downstream target gene Gli1/2 of Smo was investigated by Western Blot and immunofluorescence assay. SmoD473H mutant cell line was constructed to study the effect of B13 against drug resistance. (1) B13 had the strongest inhibitory activity against colorectal cancer cells. (2) B13 can effectively inhibit the clone formation and EdU positive rate of colon cancer cells. (3) B13 can block the cell cycle in the G2/M phase and cell apoptosis. (4) B13 has low toxicity in vivo, and its efficacy in vivo is better than that of the Vismodegib. (5) Molecular docking and BODIPY-cyclopamine experiments showed that B13 could bind to Smo protein. (6) B13 can inhibit the protein expression of Gli1, the downstream of Smo, and inhibit its entry into the nucleus. (7) B13 could inhibit the expression of Gli1 in the HEK293 cells with SmoD473H, and the molecular docking results showed that B13 could bind SmoD473H protein. B13 with the best anti-tumor activity was screened out by MTT assay. In vitro, pharmacodynamics experiments showed that B13 could effectively inhibit the proliferation and metastasis of colorectal cancer cells, induce cell cycle arrest, and induce cell apoptosis. In vivo pharmacodynamics experiments showed that B13 was superior to Vismodegib in antitumor activity and had low toxicity in vivo. Mechanism studies have shown that B13 can bind Smo protein, inhibit the expression of downstream Gli1 and its entry into the nucleus. Notably, B13 overcomes resistance caused by SmoD473H mutations.

The Hedgehog Pathway as a Therapeutic Target in Chronic Myeloid Leukemia

Despite the development of therapeutic agents that selectively target cancer cells, relapse driven by acquired drug resistance and resulting treatment failure remains a significant issue. The highly conserved Hedgehog (HH) signaling pathway performs multiple roles in both development and tissue homeostasis, and its aberrant regulation is known to drive the pathogenesis of numerous human malignancies. However, the role of HH signaling in mediating disease progression and drug resistance remains unclear. This is especially true for myeloid malignancies. The HH pathway, and in particular the protein Smoothened (SMO), has been shown to be essential for regulating stem cell fate in chronic myeloid leukemia (CML). Evidence suggests that HH pathway activity is critical for maintaining the drug-resistant properties and survival of CML leukemic stem cells (LSCs), and that dual inhibition of BCR-ABL1 and SMO may comprise an effective therapeutic strategy for the eradication of these cells in patients. This review will explore the evolutionary origins of HH signaling, highlighting its roles in development and disease, which are mediated by canonical and non-canonical HH signaling. Development of small molecule inhibitors of HH signaling and clinical trials using these inhibitors as therapeutic agents in cancer and their potential resistance mechanisms, are also discussed, with a focus on CML.