Inhibition of monocarboxylate transporter 1 has been proposed as a therapeutic approach to perturb lactate shuttling in tumor cells that lack monocarboxylate transporter 4. We examined the monocarboxylate transporter 1 inhibitor AZD3965, currently in phase I clinical studies, as a potential therapy for diffuse large B-cell lymphoma and Burkitt lymphoma. Whilst extensive monocarboxylate transporter 1 protein was found in 120 diffuse large B-cell lymphoma and 10 Burkitt lymphoma patients’ tumors, monocarboxylate transporter 4 protein expression was undetectable in 73% of the diffuse large B-cell lymphoma samples and undetectable or negligible in each Burkitt lymphoma sample. AZD3965 treatment led to a rapid accumulation of intracellular lactate in a panel of lymphoma cell lines with low monocarboxylate transporter 4 protein expression and potently inhibited their proliferation. Metabolic changes induced by AZD3965 in lymphoma cells were consistent with a feedback inhibition of glycolysis. A profound cytostatic response was also observed in vivo: daily oral AZD3965 treatment for 24 days inhibited CA46 Burkitt lymphoma growth by 99%. Continuous exposure of CA46 cells to AZD3965 for 7 weeks in vitro resulted in a greater dependency upon oxidative phosphorylation. Combining AZD3965 with an inhibitor of mitochondrial complex I (central to oxidative phosphorylation) induced significant lymphoma cell death in vitro and reduced CA46 disease burden in vivo. These data support clinical examination of AZD3965 in Burkitt lymphoma and diffuse large B-cell lymphoma patients with low tumor monocarboxylate transporter 4 expression and highlight the potential of combination strategies to optimally target the metabolic phenotype of tumors.

Neuroblastoma is the most common paediatric solid tumour and prognosis remains poor for high-risk cases despite the use of multimodal treatment. Analysis of public drug sensitivity data showed neuroblastoma lines to be sensitive to indisulam, a molecular glue that selectively targets RNA splicing factor RBM39 for proteosomal degradation via DCAF15-E3-ubiquitin ligase. In neuroblastoma models, indisulam induces rapid loss of RBM39, accumulation of splicing errors and growth inhibition in a DCAF15-dependent manner. Integrative analysis of RNAseq and proteomics data highlight a distinct disruption to cell cycle and metabolism. Metabolic profiling demonstrates metabolome perturbations and mitochondrial dysfunction resulting from indisulam. Complete tumour regression without relapse was observed in both xenograft and the Th-MYCN transgenic model of neuroblastoma after indisulam treatment, with RBM39 loss, RNA splicing and metabolic changes confirmed in vivo. Our data show that dual-targeting of metabolism and RNA splicing with anticancer indisulam is a promising therapeutic approach for high-risk neuroblastoma.

Neuroblastoma is the most common childhood extracranial solid tumor. In high-risk cases, many of which are characterized by amplification of MYCN, outcome remains poor. Mutations in the p53 (TP53) tumor suppressor are rare at diagnosis, but evidence suggests that p53 function is often impaired in relapsed, treatment-resistant disease. To address the role of p53 loss of function in the development and pathogenesis of high-risk neuroblastoma, we generated a MYCN-driven genetically engineered mouse model in which the tamoxifen-inducible p53ER TAM fusion protein was expressed from a knock-in allele (Th-MYCN/Trp53 KI ). We observed no significant differences in tumor-free survival between Th-MYCN mice heterozygous for Trp53 KI (n ¼ 188) and Th-MYCN mice with wild-type p53 (n ¼ 101). Conversely, the survival of Th-MYCN/Trp53 KI/KI mice lacking functional p53 (n ¼ 60) was greatly reduced. We found that Th-MYCN/Trp53 KI/KI tumors were resistant to ionizing radiation (IR), as expected. However, restoration of functional p53ER TAM reinstated sensitivity to IR in only 50% of Th-MYCN/ Trp53 KI/KI tumors, indicating the acquisition of additional resistance mechanisms. Gene expression and metabolic analyses indicated that the principal acquired mechanism of resistance to IR in the absence of functional p53 was metabolic adaptation in response to chronic oxidative stress. Tumors exhibited increased antioxidant metabolites and upregulation of glutathione S-transferase pathway genes, including Gstp1 and Gstz1, which are associated with poor outcome in human neuroblastoma. Accordingly, glutathione depletion by buthionine sulfoximine together with restoration of p53 activity resensitized tumors to IR. Our findings highlight the complex pathways operating in relapsed neuroblastomas and the need for combination therapies that target the diverse resistance mechanisms at play. Cancer Res; 76(10); 3025-35. Ó2016 AACR.

Background: Overexpression of EGFR is a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). Patients with HNSCC who respond to EGFR-targeted tyrosine kinase inhibitors (TKIs) eventually develop acquired resistance. Strategies to identify HNSCC patients likely to benefit from EGFR-targeted therapies, together with biomarkers of treatment response, would have clinical value.Methods: Functional MRI and 18F-FDG PET were used to visualize and quantify imaging biomarkers associated with drug response within size-matched EGFR TKI-resistant CAL 27 (CALR) and sensitive (CALS) HNSCC xenografts in vivo, and pathological correlates sought.Results: Intrinsic susceptibility, oxygen-enhanced and dynamic contrast-enhanced MRI revealed significantly slower baseline R2∗, lower hyperoxia-induced ΔR2∗ and volume transfer constant Ktrans in the CALR tumors which were associated with significantly lower Hoechst 33342 uptake and greater pimonidazole-adduct formation. There was no difference in oxygen-induced ΔR1 or water diffusivity between the CALR and CALS xenografts. PET revealed significantly higher relative uptake of 18F-FDG in the CALR cohort, which was associated with significantly greater Glut-1 expression.Conclusions: CALR xenografts established from HNSCC cells resistant to EGFR TKIs are more hypoxic, poorly perfused and glycolytic than sensitive CALS tumors. MRI combined with PET can be used to non-invasively assess HNSCC response/resistance to EGFR inhibition.

Overexpression of EGFR has been identified as a negative prognostic factor in squamous cell carcinoma of the head and neck (SCCHN). Patients with SCCHN who respond to EGFR-targeted tyrosine kinase inhibitors (TKIs) eventually develop acquired resistance. Colocalization of EGFR expression and tumor hypoxia in SCCHN is associated with poor outcome, suggesting a role for hypoxia in drug resistance. Non-invasive imaging strategies to accurately identify and monitor patients whose tumors have become resistant to EGFR-TKI therapy would have clinical value. As part of a pre-clinical multiparametric imaging study, we have investigated EGFR-TKI resistance using intrinsic susceptibility magnetic resonance imaging (IS-MRI), which relies on the dependence of the MRI transverse relaxation rate R2* on the ratio of oxy- to deoxyhemoglobin in blood. Given its relationship to blood oxygen saturation and pO2 in and around blood vessels, R2* and hyperoxia-induced ΔR2* are being investigated as imaging biomarkers of tumor oxygenation. IS-MRI was performed on size-matched xenografts derived from the EGFR TKI sensitive SCCHN cell line CAL27 and an isogenic subline resistant to multiple TKIs (gefitinib, erlotinib, and afatinib). Tumor-bearing mice were administered the hypoxia marker pimonidazole, and baseline R2* quantified from resistant (n=8) or sensitive (n=7) tumors whilst the host breathed air. Gas delivery was then switched to 100% O2, and tumour R2* measured again. Finally, mice were administered the perfusion marker Hoechst 33342. The extent of functional tumor vasculature and hypoxia were quantified ex vivo using fluorescence microscopy. Resistant tumors revealed regions of fast R2* (functional, deoxygenated vasculature) restricted primarily to the tumor periphery, while sensitive tumors exhibited a more heterogeneous distribution of fast R2* throughout. There was no significant difference in baseline R2* between the two cohorts (64 ± 4s-1 and 76 ± 5s-1, p=0.07). Hyperoxia resulted in a significantly smaller ΔR2* in the resistant tumors (-2.4 ± 1.4s-1) compared to sensitive tumors (-9.1 ± 2s-1, p<0.05). Resistant tumors had significantly lower Hoechst 33342 uptake (9.5 ± 1.3%, p<0.01) and increased pimonidazole adducts (21 ± 2%, p<0.01) compared to sensitive tumors (19.2 ± 3% and 10.4 ± 2% respectively). Tumor regions of fast R2* and hyperoxic-induced ΔR2* were spatially associated with Hoechst 33342 uptake. Tumors with resistance to EGFR-TKIs exhibited a reduced hemodynamic MRI response that was associated with decreased vessel perfusion and increased tumor hypoxia. IS-MRI informs on phenotypic differences in tumor blood vessel distribution, functionality and oxygenation associated with EGFR-TKI resistance and sensitivity, affording useful non-invasive imaging biomarkers for investigating EGFR drug resistance and tumor hypoxia. Citation Format: Lauren CJ Baker, Carol Box, Arti Sikka, Gary Box, Suzanne A. Eccles, Simon P. Robinson. Evaluating imaging biomarkers of acquired resistance to targeted EGFR therapy in xenograft models of human squamous cell carcinoma of the head and neck (SCCHN). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2013-4459