PIK3CA Mutations in Diffuse Gliomas: An Update on Molecular Stratification, Prognosis, Recurrence, and Aggressiveness

Introduction: PIK3CA is one of the most mutated oncogenes in solid tumors. In breast cancer (ER-positive, HER2-negative), these events represent a predictive biomarker of response to alpelisib. In glioblastomas (GBM), PIK3CA mutations were described as early constitutive events. Here, we investigated PIK3CA mutational profile across glioma molecular subgroups and its relevance during glioma recurrence. Furthermore, PIK3CA mutations’ effect in PI3K pathway, prognosis, and response to therapy was also explored. Material and Methods: Exons 10 and 21 of PIK3CA mutations were evaluated in 394 gliomas and 19 glioma recurrences from Instituto Português de Oncologia Lisboa Francisco Gentil (IPOLFG) and compared with The Cancer Genome Atlas (TCGA) data. TIMER2.0 and NetMHCpan4.1 were used to assess the immune-microenvironment contribution. Results: PIK3CA mutations were identified among all glioma subgroups, although with no impact on their stratification or prognosis. In both cohorts (IPOLFG and TCGA), PIK3CA mutation frequencies in IDH-mutant and IDH-wild-type GBM were similar (IPOLFG: 9% and 3%; TCGA: 8% and 2%). These mutations were not mutually exclusive with PTEN deletion and EGFR amplification. Despite their reduced frequency, we discovered PIK3CA mutations were maintained during glioma recurrence regardless of administered therapies. The immune microenvironment might not contribute to this phenotype as PIK3CA mutations did not influence immune cell infiltration. Conclusions: Despite the absence of a predominant effect in glioma stratification, PIK3CA mutations were maintained during glioma recurrence, possibly contributing to glioma cell survival, representing promising therapeutic targets in recurrent glioma. Nevertheless, understanding the potential synergistic effects between PIK3CA mutations, PTEN deletion, and EGFR amplification is pivotal to targeted therapies’ efficiency.

Cinobufagin Is a Selective Anti-Cancer Agent against Tumors with EGFR Amplification and PTEN Deletion

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor, and almost half of the patients carrying EGFR-driven tumor with PTEN deficiency are resistant to EGFR-targeted therapy. EGFR amplification and/or mutation is reported in various epithelial tumors. This series of studies aimed to identify a potent compound against EGFR-driven tumor. We screened a chemical library containing over 600 individual compounds purified from Traditional Chinese Medicine against GBM cells with EGFR amplification and found that cinobufagin, the major active ingredient of Chansu, inhibited the proliferation of EGFR amplified GBM cells and PTEN deficiency enhanced its anti-proliferation effects. Cinobufagin also strongly inhibited the proliferation of carcinoma cell lines with wild-type or mutant EGFR expression. In contrast, the compound only weakly inhibited the proliferation of cancer cells with low or without EGFR expression. Cinobufagin blocked EGFR phosphorylation and its downstream signaling, which additionally induced apoptosis and cytotoxicity in EGFR amplified cancer cells. In vivo, cinobufagin blocked EGFR signaling, inhibited cell proliferation, and elicited apoptosis, thereby suppressing tumor growth in both subcutaneous and intracranial U87MG-EGFR xenograft mouse models and increasing the median survival of nude mice bearing intracranial U87MG-EGFR tumors. Cinobufagin is a potential therapeutic agent for treating malignant glioma and other human cancers expressing EGFR.

MLN0128, a Potential Target Therapy for Myeloid Proliferation Neoplasia (MPN) with a Benefit of Prolonging Survival

MLN0128 (TAK-228, INK128) is an investigational new drug (IND) that is currently being evaluated in clinical trials and has showed promising efficacies in patients with solid cancer. MLN0128 is one of the second generation of inhibitors for mammalian target of rapamycin (mTORi), which suppresses the dysregulated cell survival signaling activities in cancer cells by specifically inhibiting both mTORC1 and mTORC2 in PI3K/mTOR pathway. MLN0128 was reported inhibiting acute myeloid leukemia (AML) stem cells and progenitors [Zeng et al, Oncotarget, 2016]. However, it is yet to be clarified whether MLN0128 can improve survival time in leukemia patients. Here, we report data from the investigation of the impact of MLN0128 on leukemia survival in mice. We tested MLN0128 in a published mouse model which simultaneously produces experimental mice with genotypes that are associated with pediatric leukemia, including myeloproliferative neoplasm (MPN) and juvenile myelomonocytic leukemia (JMML), and adult chronic myelomonocytic leukemia (CMML) [Vara et al, Blood Adv.2020]. Specifically, mice with deletion of Phosphatase and tensin homolog gene (Pten) and loss-of-heterozygosity of Neurofibromin 1 (Nf1) (Pten Δ/ΔNf1 LOH) develop JMML, mice with Pten deletion and Nf1 heterozygosity (Pten Δ/ΔNf1 Δ/wt) develop pediatric MPN, both of which cause juvenile death; mice with a single copy of Pten deletion and Nf1 LOH (Pten Δ/wtNf1 LOH) develop CMML in adulthood. We previously reported that all of those diseased mice shared a feature with elevated activities in PI3K/MAPK/mTOR pathways. We first collected nucleated cells from bone marrow (BM) of the diseased mice and tested the inhibition of MLN0128 on colony formation of hematopoietic progenitors (CFU-GM) at a concentration range of 1-1000nM. Our data demonstrated that MLN028 significantly inhibited the CFU-GM colony formation of BM cells from mice with all three diseased genotypes, in a dose-dependent fashion. Importantly, the IC50 for CFU-GM was less than 100nM, which is in the concentration range of MLN0128 in the blood from patients who orally received MLN0128 at dosage range of 4-12mg/day [Ghobrial et al AJH 2016]. We then investigated the impact of MLN0128 on life span in diseased mice. We administrated MLN0128 in gavage at dosages of 0.5-1μg/g body weight, starting at an age of postnatal day 17-18 (PND17-18), 3-5 times a week, repeated for 5 weeks followed by a pause in 6 th week every course. Our data showed that MLN0128 significantly reduced the spleen sizes in treated mice with all diseased genotypes comparing to those treated with vehicle. The survival time was significantly prolonged in MLN0128-treated mice with genotypes of MNP (n=7 vs 3, median 150 vs 102 days) and CMML (n=7 vs 6, median 100 vs 56 days), comparing with the mice treated by vehicle. Unfortunately, we did not observe a survival improvement in treated JMML mice, but substantially swollen kidneys were found in the dead JMML mice, which was likely due to tumor-lysis syndrome because JMML mice carried a significantly worse leukemic burden. In conclusion, our data demonstrate that MLN0128 can significantly inhibit colony formation of BM cells from mice with genotypes of JMML, MPN, and CMML; and MLN0128 can significantly expand survival time of mice with genotypes of MPN and CMML, which has never been reported in previous studies that were related to mTORi in pediatric leukemia. Our data suggest that MLN0128 may be a potential target therapy for pediatric MPN and CMML with a benefit of prolonging survival. Further investigation is ongoing to evaluate the potential of MLN0128 in improving survival of JMML mice after minimizing the tumor-lysis syndrome by tipping down MLN0128 dosage. Disclosures Bertaina: Cellevolve Bio: Membership on an entity's Board of Directors or advisory committees; Neovii: Membership on an entity's Board of Directors or advisory committees; AdicetBio: Membership on an entity's Board of Directors or advisory committees.

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

Identification of Androgen Receptor Metabolic Correlome Reveals the Repression of Ceramide Kinase by Androgens

Prostate cancer (PCa) is one of the most prevalent cancers in men. Androgen receptor signaling plays a major role in this disease, and androgen deprivation therapy is a common therapeutic strategy in recurrent disease. Sphingolipid metabolism plays a central role in cell death, survival, and therapy resistance in cancer. Ceramide kinase (CERK) catalyzes the phosphorylation of ceramide to ceramide 1-phosphate, which regulates various cellular functions including cell growth and migration. Here we show that activated androgen receptor (AR) is a repressor of CERK expression. We undertook a bioinformatics strategy using PCa transcriptomics datasets to ascertain the metabolic alterations associated with AR activity. CERK was among the most prominent negatively correlated genes in our analysis. Interestingly, we demonstrated through various experimental approaches that activated AR reduces the mRNA expression of CERK: (i) expression of CERK is predominant in cell lines with low or negative AR activity; (ii) AR agonist and antagonist repress and induce CERK mRNA expression, respectively; (iii) orchiectomy in wildtype mice or mice with PCa (harboring prostate-specific Pten deletion) results in elevated Cerk mRNA levels in prostate tissue. Mechanistically, we found that AR represses CERK through interaction with its regulatory elements and that the transcriptional repressor EZH2 contributes to this process. In summary, we identify a repressive mode of AR that influences the expression of CERK in PCa.

Conditional ERK3 overexpression cooperates with PTEN deletion to promote lung adenocarcinoma formation in mice

Extracellular signal-regulated kinase 3 (ERK3), also known as MAPK6, belongs to the atypical mitogen-activated protein kinases (MAPKs) subfamily. In comparison with the well-studied classical MAPKs ERK1 and ERK2, much less is known about the cellular and molecular actions of ERK3. Accumulating studies have revealed the upregulation of ERK3 expression and suggested an important role for ERK3 in promoting tumor cell growth and invasion in multiple cancers, in particular lung cancer. However, it is unknown whether or not ERK3 plays a role in spontaneous tumorigenesis. To determine the role of ERK3 in lung tumorigenesis, we created a conditional ERK3 transgenic mouse line in which ERK3 transgene expression is controlled by Cre recombinase. By crossing with a lung tissue-specific CCSP-iCre mouse line, we have found that conditional ERK3 overexpression cooperates with PTEN deletion to induce the formation of lung adenocarcinomas (LUADs). Mechanistically, ERK3 overexpression stimulates activating phosphorylations of ERBB3 and ERBB2 by upregulating SP1-mediated gene transcription of NRG1, a potent ligand for ERBB3/ERBB2. To our knowledge, our study is the first revealing a bona fide tumor-promoting role for ERK3 using genetically engineered mouse models. Together with previous findings showing important roles of ERK3 in cultured cells and in xenograft lung tumor model, our findings corroborate that ERK3 acts as an oncoprotein in promoting LUAD development and progression.

Distinct roles of tumor associated mutations in collective cell migration

Recent evidence suggests that groups of cells are more likely to form clinically dangerous metastatic tumors, emphasizing the importance of understanding mechanisms underlying collective behavior. The emergent collective behavior of migrating cell sheets in vitro has been shown to be disrupted in tumorigenic cells but the connection between this behavior and in vivo tumorigenicity remains unclear. We use particle image velocimetry to measure a multidimensional migration phenotype for genetically defined human breast epithelial cell lines that range in their in vivo behavior from non-tumorigenic to aggressively metastatic. By using cells with controlled mutations, we show that PTEN deletion enhances collective migration, while Ras activation suppresses it, even when combined with PTEN deletion. These opposing effects on collective migration of two mutations that are frequently found in patient tumors could be exploited in the development of novel treatments for metastatic disease. Our methods are based on label-free phase contrast imaging, and thus could easily be applied to patient tumor cells. The short time scales of our approach do not require potentially selective growth, and thus in combination with label-free imaging would allow multidimensional collective migration phenotypes to be utilized in clinical assessments of metastatic potential.