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Rhabdomyosarcoma (RMS) is a highly malignant soft-tissue tumor of childhood deriving from skeletal muscle cells. RMS can be classified in two major histologic subtypes: embryonal (ERMS) and alveolar (ARMS), the latter being characterized by the PAX3/7-FKHR translocation. Here we first investigated whether the Met receptor, a transcriptional target of PAX3 and PAX7, has a role in PAX3-FKHR–mediated transformation. Following PAX3-FKHR transduction, Met was up-regulated in mouse embryonal fibroblasts (MEF), NIH 3T3 and C2C12 cells, and they all acquired anchorage independence. This property was lost in low serum but addition of hepatocyte growth factor/scatter factor (HGF/SF) rescued soft-agar growth. Genetic proof that Met is necessary for this PAX3-FKHR–mediated effect was obtained by transducing with PAX3-FKHR MEFs derived from Met mutant (MetD/D) and wild-type (Met+/+) embryos. Only Met+/+ MEFs acquired anchorage-independent growth whereas PAX3-FKHR–transduced MetD/D cells were unable to form colonies in soft agar. To verify if Met had a role in RMS maintenance, we silenced the receptor by transducing ERMS and ARMS cell lines with an inducible lentivirus expressing an anti-Met short hairpin RNA (shRNA). Met down-regulation significantly affected RMS cells proliferation, survival, invasiveness, and anchorage-independent growth. Finally, induction of the Met-directed shRNA promoted a dramatic reduction of tumor mass in a xenograft model of RMS. Our data show that both ARMS- and ERMS-derived cell lines, in spite of the genetic drift which may have occurred in years of culture, seem to have retained an “addiction” to the Met oncogene and suggest that Met may represent a target of choice to develop novel therapeutic strategies for ARMS. (Cancer Res 2006; 66(9): 4742-9)

Supplementary Figure 1 from Validation of Met as a Therapeutic Target in Alveolar and Embryonal Rhabdomyosarcoma

Supplementary Figure 2 from Validation of Met as a Therapeutic Target in Alveolar and Embryonal Rhabdomyosarcoma

Multiple myeloma (MM) shows constitutive activation of canonical and non-canonical nuclear factor-ĸB (NF-ĸB) signaling through genetic mutations or stimuli from the tumour microenvironment (TME). A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-ĸB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules IL-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the mRNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on normal long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B-cells in an IL-21-dependent in vitro PC-differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody (mAb) therapies by increasing CD38-expression on tumour cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared to normal PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME.

The transcription factor β-catenin is a key player in many cellular processes, including stem cell renewal, cellular homeostasis and inflammation. Deregulation of β-catenin occurs frequently in several cancer types by mutation or overexpression of the β-catenin gene or mutation of negative regulators such as Adenomatous Polyposis Coli (APC). ST316 is a novel peptide antagonist that specifically interferes with the association of β-catenin with its co-activator BCL9, disrupting β-catenin nuclear localization and attenuating target gene expression. In an orthotopic triple-negative breast cancer (TNBC) model, ST316 demonstrates potent anti-tumor activity, resulting in 84% tumor growth inhibition (TGI) (p Citation Format: Claudio Scuoppo, Lila Ghamsari, Erin Gallagher, Siok Leong, Mark Koester, Rick Ramirez, Jerel Gonzales, Gene Merutka, Barry Kappel, Abi Vainstein-Haras, Jim Rotolo. Immunotherapeutic potential of ST316, a peptide antagonist of β-catenin [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 LB016.

CCAAT/Enhancer Binding Protein β (C/EBPβ) is a basic leucine zipper (bZIP) transcription factor that causes aberrant gene expression in many cancers. Upregulated or overactivated C/EBPβ drives oncogenesis by promoting tumor survival and proliferation and is a critical regulator of the immunosuppressive tumor microenvironment (TME). Specifically, C/EBPβ regulates macrophage differentiation, activating a transcriptional program driving macrophage polarization toward immunosuppressive M2-type myeloid-derived suppressor cells (MDSCs). Consistently, activation of C/EBPβ correlates with poor prognosis in several types of human cancer. Thus, targeting C/EBPβ to reprogram tumor-associated macrophages (TAMs) from the M2 toward the immune-promoting M1 phenotype represents an attractive strategy to enhance antitumor immunity. ST101 is a novel peptide antagonist of C/EBPβ dimerization that inhibits C/EBPβ-dependent gene expression. Here we evaluated the impact of ST101 on macrophage differentiation, cytotoxic T-cell activation, and in vivo anti-tumor activity. Primary human macrophages cultured from Peripheral Blood Mononuclear Cells (hPBMCs) were activated toward the M1 or M2 phenotype by LPS and IFNγ or IL-4, respectively. ST101 exposure dose-dependently suppressed expression of M2 markers (CD163, CD206) while inducing M1 markers (CD80, CD86) by flow cytometry and quantitative PCR, resulting in a 40-fold increase in the M1/M2 ratio without substantial impact on cell viability. Next, in co-cultures of T cells with M2 macrophage, ST101 exposure resulted in a 4-fold increase in T-cell activation compared to control M2/T cell co-cultures, as measured by intracellular IFN-γ staining. Importantly, ST101 did not suppress proliferation or activity of T cells cultured alone. Finally, in an orthotopic TNBC model in vivo, ST101 in combination with anti-PD-1 treatment enhanced anti-tumor activity compared to either single agent alone. The observed increase in tumor growth inhibition was accompanied by a reduced TAM fraction and increased intratumoral and peripheral M1/M2 ratios. ST101 is being evaluated in a Phase 1-2 clinical study in patients with advanced unresectable and metastatic solid tumors (NCT04478279). Initial gene expression analysis performed on 8 paired patient biopsies (prior to ST101 exposure and within 24 hrs of ST101 administration during cycle 2 of therapy) collected during dose escalation (4 mg/kg ST101 or greater) indicates a significant decrease in expression of multiple factors involved in M2 polarization, including CD209, SIGLEC5 and IL-24, and T cell suppression, including FoxP3 and inhibitory KIR proteins. The result is a decrease in intratumoral regulatory T cell (Treg) vs. TIL ratio, indicating a shift towards a more immunoactive TME. Overall, these results support a novel, macrophage-driven mechanism of action for ST101 as anticancer agent and suggest the exploration of ST101 in immune-oncology therapeutic strategies. Citation Format: Claudio Scuoppo, Rick Ramirez, Siok Leong, Lila Ghamsari, Gina Capiaux, Rob Michel, Steve Kaesshaefer, Gene Merutka, Barry Kappel, Abi Vainstein-Haras, Alice Bexon, Jim Rotolo. ST101, a peptide antagonist of novel I/O target CEBPβ,reprograms MDSCs and promotes an immunoactive tumor microenvironment [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 LB236.

Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of diseases in terms of cell of origin, genetics and clinical outcome. About 30% of all DLBCL patients represent an unmet clinical need as they either do not respond to the standard first line chemo-immunotherapy or recur after initial remission. DLBCL classifications based on the NMF (Non-Negative Matrix Factorization) and LymphGen algorithms have led to the identification of genetic subtypes based on the co-occurrence of specific lesions. Repositioning drugs that are approved or in active clinical development for other indications can be a powerful strategy to match these newly uncovered DLBCL subtypes to targeted therapies. We have previously shown that the screening of large panels of DLBCL cell lines representative of the genetics of the disease can facilitate the repositioning of approved drugs for biomarker-selected populations of DLBCL patients. Repositioned drugs can then be rapidly translated to the clinical use, as they have already been extensively characterized for their safety profile. As a proof of concept, we have demonstrated that Dasatinib, a Src/Abl inhibitor approved for B-cell Acute Lymphoblastic Leukemia and Chronic Myelogenous Leukemia, is highly effective in PTEN-positive DLBCLs, irrespective of their COO class (Scuoppo et al., PNAS 2019). Here we present the results of a new screening that was performed on a panel of eight cell lines (4 ABC- and 4 GCB-DLBCLs) to test the activity of 212 drugs, either approved or in advanced clinical development, for repositioning in DLBCL, followed by validation in a larger panel of 34 genetically characterized cell lines. Our results point to inhibitors of the Nicotinamide Phosphoribosyl Transferase (NAMPT) as potently active in 63% of GCB-DLBCLs. NAMPT catalyzes the conversion of Nicotinamide (NAM) to Beta-Nicotinamide Mononucleotide (Beta-NMN). This reaction is the rate-limiting step of the Nicotinamide Adenine Dinucleotide (NAD) salvage pathway, the major metabolic route of NAD regeneration in mammalian cells. We validated the on-target activity of NAMPT inhibitors by multiple genetic and pharmacological approaches. First, we found that the activities of three chemically distinct drugs (FK-866, STF-118804 and KPT-9274) were highly correlated across the 34 lines DLBCL panel. Second, we were able to abolish the activity of all three NAMPT inhibitors by supplementing cells with Beta-NMN. Third, we showed that transduction of the drug-resistant mutant NAMPT H191R offsets the activity of the drugs. Dose-response assays on the full DLBCL cell line panel confirmed ABC-DLBCL resistance and also highlighted the presence of sensitive (GCB-S) and resistant (GCB-R) GCB subsets that can be separated by a subnanomolar IC50 threshold. To generate biomarkers capable of predicting GCB-S patients, we examined the RNA-seq profiles and genetic make-ups of the DLBCL cell lines collection and observed that the GCB-S subset was associated to the LymphGen EZB subtype, characterized by the presence of EZH2 mutations and BCL2 translocations. Conversely, the GCB-R subtype was linked to a 5-gene expression signature for the Kynurenine De Novo pathway, an alternative route for NAD synthesis. Accordingly, expression of each of the five De Novo Kynurenine pathway genes induced resistance to NAMPT inhibitors. These results were validated in xenotransplants of luciferized DLBCL lines and Patient Derived Xenotransplant models (PDXs) that were transcriptionally classified for the status of the Kynurenine De Novo signature. Together, these data support the repositioning of NAMPT inhibitors as a therapeutically relevant strategy for EZB-type GCB-DLBCLs. Disclosures Pasqualucci: Astra Zeneca: Research Funding; Sanofi: Research Funding.