Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR.

Tenovin-6 is the most studied member of a family of small molecules with antitumour activity in vivo. Previously, it has been determined that part of the effects of tenovin-6 associate with its ability to inhibit SirT1 and activate p53. However, tenovin-6 has also been shown to modulate autophagic flux. Here we show that blockage of autophagic flux occurs in a variety of cell lines in response to certain tenovins, that autophagy blockage occurs regardless of the effect of tenovins on SirT1 or p53, and that this blockage is dependent on the aliphatic tertiary amine side chain of these molecules. Additionally, we evaluate the contribution of this tertiary amine to the elimination of proliferating melanoma cells in culture. We also demonstrate that the presence of the tertiary amine is sufficient to lead to death of tumour cells arrested in G1 phase following vemurafenib treatment. We conclude that blockage of autophagic flux by tenovins is necessary to eliminate melanoma cells that survive B-Raf inhibition and achieve total tumour cell kill and that autophagy blockage can be achieved at a lower concentration than by chloroquine. This observation is of great relevance as relapse and resistance are frequently observed in cancer patients treated with B-Raf inhibitors.

PTEN promoter hypermethylation is nearly universal and PTEN copy number loss occurs in ~25% of fusion-negative rhabdomyosarcoma (FN-RMS). Here we show Pten deletion in a mouse model of FN-RMS results in less differentiated tumors more closely resembling human embryonal RMS. PTEN loss activated the PI3K pathway but did not increase mTOR activity. In wild-type tumors, PTEN was expressed in the nucleus suggesting loss of nuclear PTEN functions could account for these phenotypes. Pten deleted tumors had increased expression of transcription factors important in neural and skeletal muscle development including Dbx1 and Pax7. Pax7 deletion completely rescued the effects of Pten loss. Strikingly, these Pten;Pax7 deleted tumors were no longer FN-RMS but displayed smooth muscle differentiation similar to leiomyosarcoma. These data highlight how Pten loss in FN-RMS is connected to a PAX7 lineage-specific transcriptional output that creates a dependency or synthetic essentiality on the transcription factor PAX7 to maintain tumor identity.

Prostate cancer is the second most common cancer in men, for which there are no reliable biomarkers or targeted therapies. Here we demonstrate that elevated levels of Δ133TP53β isoform characterize prostate cancers with immune cell infiltration, particularly T cells and CD163+ macrophages. These cancers are associated with shorter progression-free survival, Gleason scores ≥ 7, and an immunosuppressive environment defined by a higher proportion of PD-1, PD-L1 and colony-stimulating factor 1 receptor (CSF1R) positive cells. Consistent with this, RNA-seq of tumours showed enrichment for pathways associated with immune signalling and cell migration. We further show a role for hypoxia and wild-type p53 in upregulating Δ133TP53 levels. Finally, AUC analysis showed that Δ133TP53β expression level alone predicted aggressive disease with 88% accuracy. Our data identify Δ133TP53β as a highly accurate prognostic factor for aggressive prostate cancer.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma that histologically resembles embryonic skeletal muscle. RMS occurs throughout the body and an exclusively myogenic origin does not account for RMS occurring in sites devoid of skeletal muscle. We previously described an RMS model activating a conditional constitutively active Smoothened mutant (SmoM2) with aP2-Cre. Using genetic fate mapping, we show SmoM2 expression in Cre-expressing endothelial progenitors results in myogenic transdifferentiation and RMS. We show that endothelium and skeletal muscle within the head and neck arise from Kdr-expressing progenitors, and that hedgehog pathway activation results in aberrant expression of myogenic specification factors as a potential mechanism driving RMS genesis. These findings suggest that RMS can originate from aberrant development of non-myogenic cells.

The development of non-genotoxic therapies that activate wild-type p53 in tumors is of great interest since the discovery of p53 as a tumor suppressor. Here we report the identification of over 100 small-molecules activating p53 in cells. We elucidate the mechanism of action of a chiral tetrahydroindazole (HZ00), and through target deconvolution, we deduce that its active enantiomer (R)-HZ00, inhibits dihydroorotate dehydrogenase (DHODH). The chiral specificity of HZ05, a more potent analog, is revealed by the crystal structure of the (R)-HZ05/DHODH complex. Twelve other DHODH inhibitor chemotypes are detailed among the p53 activators, which identifies DHODH as a frequent target for structurally diverse compounds. We observe that HZ compounds accumulate cancer cells in S-phase, increase p53 synthesis, and synergize with an inhibitor of p53 degradation to reduce tumor growth in vivo. We, therefore, propose a strategy to promote cancer cell killing by p53 instead of its reversible cell cycle arresting effect.

Malignant melanoma is the most dangerous type of skin cancer. Although recent progress in treatment has been achieved, lack of response, drug resistance and relapse remain major problems. The tumor suppressor p53 is rarely mutated in melanoma, yet it is inactive in the majority of cases due to dysregulation of upstream pathways. Thus, we screened for compounds that can activate p53 in melanoma cells. Here we describe effects of the small molecule MJ25 (2-{[2-(1,3-benzothiazol-2-ylsulfonyl)ethyl]thio}-1,3-benzoxazole), which increased the level of p53-dependent transactivation both as a single agent and in combination with nutlin-3. Furthermore, MJ25 showed potent cytotoxicity towards melanoma cell lines, whilst having weaker effects against human normal cells. MJ25 was also identified in an independent screen as an inhibitor of thioredoxin reductase 1 (TrxR1), an important selenoenzyme in the control of oxidative stress and redox regulation. The well-characterized TrxR inhibitor auranofin, which is FDA-approved and currently in clinical trials against leukemia and a number of solid cancers, displayed effects comparable with MJ25 on cells and led to eradication of cultured melanoma cells at low micromolar concentrations. In conclusion, auranofin, MJ25 or other inhibitors of TrxR1 should be evaluated as candidate compounds or leads for targeted therapy of malignant melanoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.