Targeting Mutant p53 for Cancer Treatment: Moving Closer to Clinical Use?

Mutant p53 is one of the most attractive targets for new anti-cancer drugs. Although traditionally regarded as difficult to drug, several new strategies have recently become available for targeting the mutant protein. One of the most promising of these involves the use of low molecular weight compounds that promote refolding and reactivation of mutant p53 to its wild-type form. Several such reactivating drugs are currently undergoing evaluation in clinical trials, including eprenetapopt (APR-246), COTI-2, arsenic trioxide and PC14586. Of these, the most clinically advanced for targeting mutant p53 is eprenetapopt which has completed phase I, II and III clinical trials, the latter in patients with mutant TP53 myelodysplastic syndrome. Although no data on clinical efficacy are currently available for eprenetapopt, preliminary results suggest that the drug is relatively well tolerated. Other strategies for targeting mutant p53 that have progressed to clinical trials involve the use of drugs promoting degradation of the mutant protein and exploiting the mutant protein for the development of anti-cancer vaccines. With all of these ongoing trials, we should soon know if targeting mutant p53 can be used for cancer treatment. If any of these trials show clinical efficacy, it may be a transformative development for the treatment of patients with cancer since mutant p53 is so prevalent in this disease.

Statins inhibit proliferation and induce apoptosis in triple-negative breast cancer cells.

TP53 (p53) is mutated in 80-90% of cases of triple-negative breast cancer (TNBC). Statins, which are widely used to treat elevated cholesterol, have recently been shown to degrade mutant p53 protein and exhibit anti-cancer activity. The aim of this work was to evaluate the potential of statins in the treatment of TNBC. The anti-proliferative effects of 2 widely used statins were investigated on a panel of 15 cell lines representing the different molecular subtypes of breast cancer. Significantly lower IC50 values were found in triple-negative (TN) than in non-TN cell lines (atorvastatin, p < 0.01; simvastatin p < 0.05) indicating greater sensitivity. Furthermore, cell lines containing mutant p53 were more responsive to both statins than cell lines expressing wild-type p53, suggesting that the mutational status of p53 is a potential predictive biomarker for statin response. In addition to inhibiting proliferation, simvastatin was also found to promote cell cycle arrest and induce apoptosis. Using an apoptosis array capable of detecting 43 apoptosis-associated proteins, a novel protein shown to be upregulated by simvastatin was the IGF-signalling modulator, IGBP4, a finding we confirmed by Western blotting. Finally, we found synergistic growth inhibition between simvastatin and the IGF-1R inhibitor, OSI-906 as well as between simvastatin and doxorubicin or docetaxel. Our work suggests repurposing of statins for clinical trials in patients with TNBC. Based on our findings, we suggest that these trials investigate statins in combination with either doxorubicin or docetaxel and include p53 mutational status as a potential predictive biomarker.

MYC as a therapeutic target for the treatment of triple-negative breast cancer: preclinical investigations with the novel MYC inhibitor, MYCi975.

BACKGROUND: MYC is one of the most frequently altered driver genes in triple-negative breast cancer (TNBC). The aim of this study was to evaluate targeting MYC for the treatment of TNBC. METHODS: The anti-proliferative and apoptosis-inducing effects of the recently discovered MYC inhibitor, MYCi975 were investigated in a panel of 14 breast cancer cell lines representing the main molecular forms of breast cancer. RESULTS: IC50 values for growth inhibition by MYCi975 varied from 2.49 to 7.73 µM. Response was inversely related to endogenous MYC levels as measured by western blotting (p = 0.047, r = - 0.5385) or ELISA (p = 0.001, r = - 0.767), i.e., response to MYCi975 decreased as endogenous MYC levels increased. MYCi975 also induced variable levels of apoptosis across the panel of cell lines, ranging from no detectable induction to 80% induction. Inhibition of proliferation and induction of apoptosis were greater in TNBC than in non-TNBC cell lines (p = 0.041 and p = 0.001, respectively). Finally, combined treatment with MYCi975 and either paclitaxel or doxorubicin resulted in enhanced cell growth inhibition. DISCUSSION: Our findings open the possibility of targeting MYC for the treatment of TNBC. Based on our results, we suggest that trials use a combination of MYCi975 and either docetaxel or doxorubicin and include MYC as a putative therapy predictive biomarker.

Targeting p53 for the treatment of cancer.

Dysfunction of the TP53 (p53) gene occurs in most if not all human malignancies. Two principal mechanisms are responsible for this dysfunction; mutation and downregulation of wild-type p53 mediated by MDM2/MDM4. Because of its almost universal inactivation in malignancy, p53 is a highly attractive target for the development of new anticancer drugs. Although multiple strategies have been investigated for targeting dysfunctional p53 for cancer treatment, only 2 of these have so far yielded compounds for testing in clinical trials. These strategies include the identification of compounds for reactivating the mutant form of p53 back to its wild-type form and compounds for inhibiting the interaction between wild-type p53 and MDM2/MDM4. Currently, multiple p53-MDM2/MDM4 antagonists are undergoing clinical trials, the most advanced being idasanutlin which is currently undergoing testing in a phase III clinical trial in patients with relapsed or refractory acute myeloid leukemia. Two mutant p53-reactivating compounds have progressed to clinical trials, i.e., APR-246 and COTI-2. Although promising data has emerged from the testing of both MDM2/MDM4 inhibitors and mutant p53 reactivating compounds in preclinical models, it is still unclear if these agents have clinical efficacy. However, should any of the compounds currently being evaluated in clinical trials be shown to have efficacy, it is likely to usher in a new era in cancer treatment, especially as p53 dysfunction is so prevalent in human cancers.

A time-course Raman spectroscopic analysis of spontaneous in vitro microcalcifications in a breast cancer cell line.

Microcalcifications are early markers of breast cancer and can provide valuable prognostic information to support clinical decision-making. Current detection of calcifications in breast tissue is based on X-ray mammography, which involves the use of ionizing radiation with potentially detrimental effects, or MRI scans, which have limited spatial resolution. Additionally, these techniques are not capable of discriminating between microcalcifications from benign and malignant lesions. Several studies show that vibrational spectroscopic techniques are capable of discriminating and classifying breast lesions, with a pathology grade based on the chemical composition of the microcalcifications. However, the occurrence of microcalcifications in the breast and the underlying mineralization process are still not fully understood. Using a previously established model of in vitro mineralization, the MDA-MB-231 human breast cancer cell line was induced using two osteogenic agents, inorganic phosphate (Pi) and ß-glycerophosphate (ßG), and direct monitoring of the mineralization process was conducted using Raman micro-spectroscopy. MDA-MB-231 cells cultured in a medium supplemented with Pi presented more rapid mineralization (by day 3) than cells exposed to ßG (by day 11). A redshift of the phosphate stretching peak for cells supplemented with ßG revealed the presence of different precursor phases (octacalcium phosphate) during apatite crystal formation. These results demonstrate that Raman micro-spectroscopy is a powerful tool for nondestructive analysis of mineral species and can provide valuable information for evaluating mineralization dynamics and any associated breast cancer progression, if utilized in pathological samples.

MYC as a target for cancer treatment.

The MYC gene which consists of 3 paralogs, C-MYC, N-MYC and L-MYC, is one of the most frequently deregulated driver genes in human cancer. Because of its high prevalence of deregulation and its causal role in cancer formation, maintenance and progression, targeting MYC is theoretically an attractive strategy for treating cancer. As a potential anticancer target, MYC was traditionally regarded as undruggable due to the absence of a suitable pocket for high-affinity binding by low molecular weight inhibitors. In recent years however, several compounds that directly or indirectly inhibit MYC have been shown to have anticancer activity in preclinical tumor models. Amongst the most detailed investigated strategies for targeting MYC are inhibition of its binding to its obligate interaction partner MAX, prevention of MYC expression and blocking of genes exhibiting synthetic lethality with overexpression of MYC. One of the most extensively investigated MYC inhibitors is a peptide/mini-protein known as OmoMYC. OmoMYC, which acts by blocking the binding of all 3 forms of MYC to their target promoters, has been shown to exhibit anticancer activity in a diverse range of preclinical models, with minimal side effects. Based on its broad efficacy and limited toxicity, OmoMYC is currently being developed for evaluation in clinical trials. Although no compound directly targeting MYC has yet progressed to clinical testing, APTO-253, which partly acts by decreasing expression of MYC, is currently undergoing a phase I clinical trial in patients with relapsed/refractory acute myeloid leukemia or myelodysplastic syndrome.

Calcium transport and signalling in breast cancer: Functional and prognostic significance.

Comprised of a complex network of numerous intertwining pathways, the Ca2+ signalling nexus is an essential mediator of many normal cellular activities. Like many other such functions, the normal physiological activity of Ca2+ signalling is frequently co-opted and reshaped in cases of breast cancer, creating a potent oncogenic drive within the affected cell population. Such modifications can occur within pathways mediating either Ca2+ import (e.g. TRP channels, ORAI-STIM1) or Ca2+ export (e.g. PMCA), indicating that both increases and decreases within cellular Ca2+ levels have the potential to increase the malignant potential of a cell. Increased understanding of these pathways may offer clinical benefit in terms of both prognosis and treatment; patient survival has been linked to expression levels of certain Ca2+ transport proteins, whilst selective targeting of these factors with novel anti-cancer agents has demonstrated a variety of anti-tumour effects in in vitro studies. In addition, the activity of several Ca2+ signalling pathways has been shown to influence chemotherapy response, suggesting that a synergistic approach coupling traditional chemotherapy with Ca2+ targeting agents may also improve patient outcome. As such, targeted modulation of these pathways represents a novel approach in precision medicine and breast cancer therapy.

The novel low molecular weight MYC antagonist MYCMI-6 inhibits proliferation and induces apoptosis in breast cancer cells.

Background The MYC oncogene is one of the most frequently altered driver genes in cancer. MYC is thus a potential target for cancer treatment as well as a biomarker for the disease. However, as a target for treatment, MYC has traditionally been regarded as "undruggable" or difficult to target. We set out to evaluate the efficacy of a novel MYC inhibitor known as MYCMI-6, which acts by preventing MYC from interacting with its cognate partner MAX. Methods MYCMI-6 response was assessed in a panel of breast cancer cell lines using MTT assays and flow cytometry. MYC gene amplification, mRNA and protein expression was analysed using the TCGA and METABRIC databases. Results MYCMI-6 inhibited cell growth in breast cancer cell lines with IC50 values varying form 0.3 µM to >10 µM. Consistent with its ability to decrease cell growth, MYCMI-6 was found to induce apoptosis in two cell lines in which growth was inhibited but not in two cell lines that were resistant to growth inhibition. Across all breast cancers, MYC was found to be amplified in 15.3% of cases in the TCGA database and 26% in the METABRIC database. Following classification of the breast cancers by their molecular subtypes, MYC was most frequently amplified and exhibited highest expression at both mRNA and protein level in the basal subtype. Conclusions Based on these findings, we conclude that for patients with breast cancer, anti-MYC therapy is likely to be most efficacious in patients with the basal subtype.

Deposition of calcium in an in vitro model of human breast tumour calcification reveals functional role for ALP activity, altered expression of osteogenic genes and dysregulation of the TRPM7 ion channel.

Microcalcifications are vital mammographic indicators contributing to the early detection of up to 50% of non-palpable tumours and may also be valuable as prognostic markers. However, the precise mechanism by which they form remains incompletely understood. Following development of an in vitro model using human breast cancer cells lines cultured with a combination of mineralisation-promoting reagents, analysis of calcium deposition, alkaline phosphatase (ALP) activity and changes in expression of key genes was used to monitor the calcification process. Two cell lines were identified as successfully mineralising in vitro, MDA-MB-231 and SKBR3. Mineralising cell lines displayed higher levels of ALP activity that was further increased by addition of mineralisation promoting media. qPCR analysis revealed changes in expression of both pro- (RUNX2) and anti- (MGP, ENPP1) mineralisation genes. Mineralisation was suppressed by chelation of intracellular Ca2+ and inhibition of TRPM7, demonstrating a functional role for the channel in formation of microcalcifications. Increased Mg2+ was also found to effectively reduce calcium deposition. These results expand the number of human breast cancer cell lines with a demonstrated in vitro mineralisation capability, provide further evidence for the role of an active, cellular process of microcalcification formation and demonstrate for the first time a role for TRPM7 mediated Ca2+ transport.

The role of DNA repair pathways in cisplatin resistant lung cancer.

Platinum chemotherapeutic agents such as cisplatin are currently used in the treatment of various malignancies such as lung cancer. However, their efficacy is significantly hindered by the development of resistance during treatment. While a number of factors have been reported that contribute to the onset of this resistance phenotype, alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon. The mode of action of cisplatin has been linked to its ability to crosslink purine bases on the DNA, thereby interfering with DNA repair mechanisms and inducing DNA damage. Following DNA damage, cells respond by activating a DNA-damage response that either leads to repair of the lesion by the cell thereby promoting resistance to the drug, or cell death via activation of the apoptotic response. Therefore, DNA repair is a vital target to improving cancer therapy and reduce the resistance of tumour cells to DNA damaging agents currently used in the treatment of cancer patients. To date, despite the numerous findings that differential expression of components of the various DNA repair pathways correlate with response to cisplatin, translation of such findings in the clinical setting are still warranted. The identification of alterations in specific proteins and pathways that contribute to these unique DNA repair pathways in cisplatin resistant cancer cells may potentially lead to a renewed interest in the development of rational novel therapies for cisplatin resistant cancers, in particular, lung cancer.