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The functions of the proto-oncoprotein c-Myc and the tumor suppressor p53 in controlling cell survival and proliferation are inextricably linked as “Yin and Yang” partners in normal cells to maintain tissue homeostasis: c-Myc induces the expression of ARF tumor suppressor (p14 ARF in human and p19 ARF in mouse) that binds to and inhibits mouse double minute 2 homolog (MDM2) leading to p53 activation, whereas p53 suppresses c-Myc through a combination of mechanisms involving transcriptional inactivation and microRNA-mediated repression. Nonetheless, the regulatory interactions between c-Myc and p53 are not retained by cancer cells as is evident from the often-imbalanced expression of c-Myc over wildtype p53. Although p53 repression in cancer cells is frequently associated with the loss of ARF, we disclose here an alternate mechanism whereby c-Myc inactivates p53 through the actions of the c-Myc-Inducible Long noncoding RNA Inactivating P53 (MILIP). MILIP functions to promote p53 polyubiquitination and turnover by reducing p53 SUMOylation through suppressing tripartite-motif family-like 2 (TRIML2). MILIP upregulation is observed amongst diverse cancer types and is shown to support cell survival, division and tumourigenicity. Thus our results uncover an inhibitory axis targeting p53 through a pan-cancer expressed RNA accomplice that links c-Myc to suppression of p53. c-Myc and p53 operate in a negative feedback manner to maintain cellular homeostasis. Here, the authors report a long noncoding RNA, MILIP as a downstream target of c-Myc and that MILIP represses p53 to support tumorigenicity.

Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography–tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, p hospho- h eavy-l a beled-spiketide FAIM S st e pped-CV D DA (pHASED), that employs online phosphoproteome deconvolution using high- f ield a symmetric waveform i on m obility s pectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients.

Organoid technology has provided unique insights into human organ development, function, and diseases. Patient-derived organoids are increasingly used for drug screening, modeling rare disorders, designing regenerative therapies, and understanding disease pathogenesis. However, the use of Matrigel to grow organoids represents a major challenge in the clinical translation of organoid technology. Matrigel is a poorly defined mixture of extracellular matrix proteins and growth factors extracted from the Engelbreth–Holm–Swarm mouse tumor. The extracellular matrix is a major driver of multiple cellular processes and differs significantly between tissues as well as in healthy and disease states of the same tissue. Therefore, we envisioned that the extracellular matrix derived from a native healthy tissue would be able to support organoid growth akin to organogenesis in vivo. Here, we have developed hydrogels from decellularized human and bovine endometrium. These hydrogels supported the growth of mouse and human endometrial organoids, which was comparable to Matrigel. Organoids grown in endometrial hydrogels were proteomically more similar to the native tissue than those cultured in Matrigel. Proteomic and Raman microspectroscopy analyses showed that the method of decellularization affects the biochemical composition of hydrogels and, subsequently, their ability to support organoid growth. The amount of laminin in hydrogels correlated with the number and shape of organoids. We also demonstrated the utility of endometrial hydrogels in developing solid scaffolds for supporting highthroughput, cell culture–based applications. In summary, endometrial hydrogels overcome a major limitation of organoid technology and greatly expand the applicability of organoids to understand endometrial biology and associated pathologies.

Breast cancer is the leading cause of cancer death in women. The majority of these deaths are due to disease metastasis, in which cancer cells disseminate to multiple organs and disrupt vital physiological functions. It is widely accepted that breast cancer cells secrete extracellular vesicles (EVs), which contain dynamic molecular cargo that act as versatile mediators of intercellular communication. Therefore, Evs. secreted by breast cancer cells could be involved in the development of metastatic disease and resistance to treatment. Moreover, changes in EV cargo could reflect the effects of therapy on their parent tumor cells. The aim of this feasibility study was to quantitatively profile the proteomes of Evs. isolated from blood samples taken from treatment sensitive and resistant metastatic breast cancer patients to identify proteins associated with responses. Three serial blood samples were collected from three patients with metastatic breast cancer receiving systemic therapy including a responder, a non-responder, and a mixed-responder. Evs. were isolated from plasma using size exclusion chromatography and their protein cargo was prepared for tandem mass tag (TMT)-labelling and quantitative analyses using two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. After filtering, we quantitatively identified 286 proteins with high confidence using a q value of 0.05. Of these, 149 were classified as EV associated candidate proteins and 137 as classical, high abundant plasma proteins. After comparing EV protein abundance between the responder and non-responder, we identified 35 proteins with unique de-regulated abundance patterns that was conserved at multiple time points. We propose that this proof-of-concept approach can be used to identify proteins which have potential as predictors of metastatic breast cancer response to treatment.

Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.

Diffuse midline glioma (DMG), including tumors diagnosed in the brainstem (diffuse intrinsic pontine glioma – DIPG), are uniformly fatal brain tumors that lack effective pharmacological treatment. Analysis of pooled CRISPR-Cas9 loss-of-function gene deletion screen datasets, identified PIK3CA and MTOR as targetable molecular dependencies across DIPG patient derived models, highlighting the therapeutic potential of the blood-brain barrier penetrant PI3K/Akt/mTOR inhibitor paxalisib. At the human equivalent maximum tolerated dose, mice treated with paxalisib experienced systemic feedback resulting in increased blood glucose and insulin levels, commensurate with DIPG patients in Phase 1b clinical trials who experienced hyperglycemia/hyperinsulinemia. To exploit genetic dependences, but maintain compliance and benefit, we optimized a paxalisib treatment regimen that employed reduced dosing more frequently, in combination with the anti-hyperglycemic drug, metformin. Combining optimized dosing with metformin restored glucose homeostasis and decreased phosphorylation of the insulin receptor in vivo, a common mechanism of PI3K-inhibitor resistance, extending the survival of DIPG xenograft models. RNA sequencing and phosphoproteomic profiling of DIPG models treated with paxalisib identified increased calcium-activated PKC signaling. Using the brain penetrant PKC inhibitor, enzastaurin in combination with paxalisib, we synergistically extended the survival of orthotopic xenograft models, benefits further promoted by metformin; thus, identifying a clinically relevant DIPG combinatorial approach. Diffuse intrinsic pontine glioma is a lethal childhood brain tumor. Here we identify PIK3CA as a genetic dependency targeted by the brain penetrant pan-PI3K-inhibitor paxalisib.