Search results

Strength in numbers: cell penetrating peptide clusters to build next-generation therapeutics

Targeting hypoxia regulated sodium driven bicarbonate transporters reduces triple negative breast cancer metastasis.

Regions of low oxygen (hypoxia) are found in >50% of breast tumours, most frequently in the more aggressive triple negative breast cancer subtype (TNBC). Metastasis is the cause of 90% of breast cancer patient deaths. Regions of tumour hypoxia tend to be more acidic and both hypoxia and acidosis increase tumour metastasis. In line with this the metastatic process is dependent on pH regulatory mechanisms. We and others have previously identified increased hypoxic expression of Na+ driven bicarbonate transporters (NDBTs) as a major mechanism of tumour pH regulation. Hypoxia induced the expression of NDBTs in TNBC, most frequently SLC4A4 and SLC4A5. NDBT inhibition (S0859) and shRNA knockdown suppressed migration (40% reduction) and invasion (70% reduction) in vitro. Tumour xenograft metastasis in vivo was significantly reduced by NDBT knockdown. To investigate the mechanism by which NDBTs support metastasis, we investigated their role in regulation of phospho-signalling, epithelial-to-mesenchymal transition (EMT) and metabolism. NDBT knockdown resulted in an attenuation in hypoxic phospho-signalling activation; most notably LYN (Y397) reduced by 75%, and LCK (Y394) by 72%. The metastatic process is associated with EMT. We showed that NDBT knockdown inhibited EMT, modulating the expression of key EMT transcription factors and ablating the expression of vimentin whilst increasing the expression of E-cadherin. NDBT knockdown also altered metabolic activity reducing overall ATP and extracellular lactate levels. These results demonstrate that targeting hypoxia-induced NDBT can be used as an approach to modulate phospho-signalling, EMT, and metabolic activity and reduce tumour migration, invasion, and metastasis in vivo.

Comprehensive Imaging Characterization of Colorectal Liver Metastases.

Colorectal liver metastases (CRLM) have heterogenous histopathological and immunohistochemical phenotypes, which are associated with variable responses to treatment and outcomes. However, this information is usually only available after resection, and therefore of limited value in treatment planning. Improved techniques for in vivo disease assessment, which can characterise the variable tumour biology, would support further personalization of management strategies. Advanced imaging of CRLM including multiparametric MRI and functional imaging techniques have the potential to provide clinically-actionable phenotypic characterisation. This includes assessment of the tumour-liver interface, internal tumour components and treatment response. Advanced analysis techniques, including radiomics and machine learning now have a growing role in assessment of imaging, providing high-dimensional imaging feature extraction which can be linked to clinical relevant tumour phenotypes, such as a the Consensus Molecular Subtypes (CMS). In this review, we outline how imaging techniques could reproducibly characterize the histopathological features of CRLM, with several matched imaging and histology examples to illustrate these features, and discuss the oncological relevance of these features. Finally, we discuss the future challenges and opportunities of CRLM imaging, with a focus on the potential value of advanced analytics including radiomics and artificial intelligence, to help inform future research in this rapidly moving field.

Partial Breast Reconstruction with Lateral Chest Wall Perforator Flap to Facilitate Breast Conservation in Breast Cancer: First 100 Cases with Cancer Outcomes at 8 Years Follow-Up and the Lessons Learned.

BACKGROUND: This is a prospective cohort study of partial breast reconstruction (PBR) with a lateral chest wall perforator flap (LCWPF) to facilitate breast conservation surgery (BCS) for women undergoing surgery for breast cancer. The study was undertaken to study the clinical and cancer outcomes. METHODS: Patients diagnosed with ductal carcinoma in situ (DCIS) or breast cancer who consented to undergo BCS with PBR with LCWPF were included in the study. A prospective database has been maintained to collect information on clinico-pathological features, complications, and follow-up. Patients were asked to complete an anonymised PROM questionnaire over the years. The hospital electronic records were interrogated for women who have completed 5 years follow-up to assess for development of recurrence/events. RESULTS: A total of 105 patients underwent PBR with LCWPFs between 2011 and 2018. Of these, 74% underwent cancer resection and PBR as one operation whilst 26% underwent PBR as a two-stage approach. The median tumor size on pre-op imaging was 30 mm for the one-stage approach and 39.5 mm for the two-stage approach (p-value=0.003). The complication rates were low and the re-operation rate for close margins was 10%, with 4% eventually requiring mastectomy. Good-to-excellent esthetic outcomes were reported in more than 80% of cases by patients and clinicians. The local recurrence rate (LR) was 2%, distant recurrence rate 10.5%, disease free survival (DFS) 86%, distant disease-free survival (DDFS) 89% and overall survival (OS) 94.8% at 4.5 years median follow-up. This procedure provides an effective oncological approach, avoiding mastectomy with a good-to-excellent cosmetic outcome. The follow-up data establishes the safety of this approach. DISCUSSION: This is the first published series of recurrence and survival data in patients undergoing PBR. We intend to continue with data collection to assess long-term outcomes beyond 10 years. The authors would recommend consideration of this technique to facilitate BCS and avoid mastectomy. REGISTRATION: Not applicable.

Low cytoplasmic NUB1 protein exerts hypoxic cell death with poorer prognosis in oestrogen receptor negative breast cancer patients.

Current prognostic biomarkers fall short in stratifying Oestrogen receptor (ER)-negative breast cancer patients regarding tumour progression risk at diagnosis. The role of AIPL1 in activating its tumour suppressor client protein, NEDD8 Ultimate Buster-1 (NUB1) remains unknown in cancer. Our study demonstrated how downregulated AIPL1 results in the deactivated NUB1 protein under hypoxic conditions. We examined the AIPL1-NUB1 pathwayin vitro using cell lines i.e. MCF-7, MDA-MB-231, RCC4 etc. NUB1 expression was assessed using Oncomine, and cBioPortal was performed to assess NUB1's prognostic significance in human cancers. In the John Radcliffe Hospital cohort (n = 122), immunohistochemistry analysis revealed downregulated AIPL1 (Log2 fold change=-0.28; p < 0.001) and upregulated NUB1 transcripts (Log2 fold change=0.59; p < 0.001) compared to adjacent normal tissues. In severe chronic hypoxia, multimerised AIPL1 localisedin the cytoplasm while NUB1 protein migrated to the nucleus, where the absence of NUB1 nuclear localisation led to cell cycle arrest. Biopsies showed that patients with lower cytoplasmic NUB1 expression (n = 57) had poorer overall survival compared to those with higher cytoplasmic expression (n = 57), HR=1.78; 95 % CI=1.01-3.35, p = 0.048. Low NUB1 protein levels in both normoxic and hypoxic conditions were associated with cell cycle arrest and upregulation ofp21 and p27 in breast cancer cell lines, correlating significantly withpoorer survival outcomes in all breast cancer and ER-negative breast cancer patients.

Proline synthesis through PYCR1 is required to support cancer cell proliferation and survival in oxygen-limiting conditions.

The demands of cancer cell proliferation alongside an inadequate angiogenic response lead to insufficient oxygen availability in the tumor microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing potential produced through tricarboxylic acid (TCA) cycle activity and mitochondrial respiration are functionally linked. As the oxidizing activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidizing NADH with the synthesis of proline as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting continued TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

Molecular insights into the stimulation of SNM1A nuclease activity by CSB during interstrand crosslink processing.

KBTBD4 Cancer Hotspot Mutations Drive Neomorphic Degradation of HDAC1/2 Corepressor Complexes.

Modular and automated synthesis of oligonucleotide-small molecule conjugates for cathepsin B mediated traceless release of payloads.

The reversible attachment of small molecules to oligonucleotides provides versatile tools for the development of improved oligonucleotide therapeutics. However, cleavable linkers in the oligonucleotide field are scarce, particularly with respect to the requirement for traceless release of the payload in vivo. Herein, we describe a cathepsin B-cleavable dipeptide phosphoramidite, Val-Ala(NB) for the automated synthesis of oligonucleotide-small molecule conjugates. Val-Ala(NB) was protected by a photolabile 2-nitrobenzyl group to improve the stability of the peptide linker during DNA synthesis. Intracellular cathepsin B digests the dipeptide efficiently, releasing the payload-phosphate which is converted to the free payload by endogenous phosphatase enzymes. With the advantages of modular synthesis and stimuli-responsive drug release, we believe Val-Ala(NB) will be a potentially valuable cleavable linker for use in oligonucleotide-drug conjugates.

Synthesis, Biophysical and Biological Evaluation of Splice-Switching Oligonucleotides with Multiple LNA-Phosphothiotriester Backbones.

Polyanionic antisense oligonucleotides hold great promise as RNA targeting drugs but issues with bioavailability hinder their development. Uncharged phosphorus-based backbones are promising alternatives but robust methods to produce them are limited. We report the synthesis and properties of oligonucleotides containing charge-neutral LNA alkyl phosphothiotriester backbones combined with 2'-O-methyl phosphorothioate nucleotides for therapeutic applications. The nature of the triester alkyl group dictates the success of solid-phase synthesis; tertiary alkyl groups are lost during the P(III) oxidation step, whereas primary alkyl groups are partially cleaved during deprotection. In contrast, oligonucleotides containing secondary phosphothiotriester linkages are stable, and large numbers of triesters can be incorporated. The modified oligonucleotides have excellent duplex stability with complementary RNA and exhibit strong nuclease resistance. To expand synthetic flexibility, oligonucleotides containing multiple internal alkynyl phosphothiotriesters can be conjugated to lipids, carbohydrates, or small molecules through CuAAC click chemistry. Oligonucleotides containing LNA-THP phosphothiotriesters exhibit high levels of pre-mRNA splice switching in eukaryotic cells.

Targeting PI3K-gamma in myeloid driven tumour immune suppression: A Systematic Review and Meta-Analysis of the Preclinical Literature

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Cyclin F-EXO1 axis controls cell cycle-dependent execution of double-strand break repair.

Ubiquitination is a crucial posttranslational modification required for the proper repair of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). DSBs are mainly repaired through homologous recombination (HR) when template DNA is present and nonhomologous end joining (NHEJ) in its absence. In addition, microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) provide backup DSBs repair pathways. However, the mechanisms controlling their use remain poorly understood. By using a high-resolution CRISPR screen of the ubiquitin system after IR, we systematically uncover genes required for cell survival and elucidate a critical role of the E3 ubiquitin ligase SCFcyclin F in cell cycle-dependent DSB repair. We show that SCFcyclin F-mediated EXO1 degradation prevents DNA end resection in mitosis, allowing MMEJ to take place. Moreover, we identify a conserved cyclin F recognition motif, distinct from the one used by other cyclins, with broad implications in cyclin specificity for cell cycle control.

Stratification to Neoadjuvant Radiotherapy in Rectal Cancer by Regimen and Transcriptional Signatures.

UNLABELLED: Response to neoadjuvant radiotherapy (RT) in rectal cancer has been associated with immune and stromal features that are captured by transcriptional signatures. However, how such associations perform across different chemoradiotherapy regimens and within individual consensus molecular subtypes (CMS) and how they affect survival remain unclear. In this study, gene expression and clinical data of pretreatment biopsies from nine cohorts of primary rectal tumors were combined (N = 826). Exploratory analyses were done with transcriptomic signatures for the endpoint of pathologic complete response (pCR), considering treatment regimen or CMS subtype. Relevant findings were tested for overall survival and recurrence-free survival. Immune and stromal signatures were strongly associated with pCR and lack of pCR, respectively, in RT and capecitabine (Cap)/5-fluorouracil (5FU)-treated patients (N = 387), in which the radiosensitivity signature (RSS) showed the strongest association. Upon addition of oxaliplatin (Ox; N = 123), stromal signatures switched direction and showed higher chances to achieve pCR than without Ox (p for interaction 0.02). Among Cap/5FU patients, most signatures performed similarly across CMS subtypes, except cytotoxic lymphocytes that were associated with pCR in CMS1 and CMS4 cases compared with other CMS subtypes (p for interaction 0.04). The only variables associated with survival were pCR and RSS. Although the frequency of pCR across different chemoradiation regimens is relatively similar, our data suggest that response rates may differ depending on the biological landscape of rectal cancer. Response to neoadjuvant RT in stroma-rich tumors may potentially be improved by the addition of Ox. RSS in preoperative biopsies provides predictive information for response specifically to neoadjuvant RT with 5FU. SIGNIFICANCE: Rectal cancers with stromal features may respond better to RT and 5FU/Cap with the addition of Ox. Within patients not treated with Ox, high levels of cytotoxic lymphocytes associate with response only in immune and stromal tumors. Our analyses provide biological insights about the outcome by different radiotherapy regimens in rectal cancer.

Identification and validation of a machine learning model of complete response to radiation in rectal cancer reveals immune infiltrate and TGFβ as key predictors.

BACKGROUND: It is uncertain which biological features underpin the response of rectal cancer (RC) to radiotherapy. No biomarker is currently in clinical use to select patients for treatment modifications. METHODS: We identified two cohorts of patients (total N = 249) with RC treated with neoadjuvant radiotherapy (45Gy/25) plus fluoropyrimidine. This discovery set included 57 cases with pathological complete response (pCR) to chemoradiotherapy (23%). Pre-treatment cancer biopsies were assessed using transcriptome-wide mRNA expression and targeted DNA sequencing for copy number and driver mutations. Biological candidate and machine learning (ML) approaches were used to identify predictors of pCR to radiotherapy independent of tumour stage. Findings were assessed in 107 cases from an independent validation set (GSE87211). FINDINGS: Three gene expression sets showed significant independent associations with pCR: Fibroblast-TGFβ Response Signature (F-TBRS) with radioresistance; and cytotoxic lymphocyte (CL) expression signature and consensus molecular subtype CMS1 with radiosensitivity. These associations were replicated in the validation cohort. In parallel, a gradient boosting machine model comprising the expression of 33 genes generated in the discovery cohort showed high performance in GSE87211 with 90% sensitivity, 86% specificity. Biological and ML signatures indicated similar mechanisms underlying radiation response, and showed better AUC and p-values than published transcriptomic signatures of radiation response in RC. INTERPRETATION: RCs responding completely to chemoradiotherapy (CRT) have biological characteristics of immune response and absence of immune inhibitory TGFβ signalling. These tumours may be identified with a potential biomarker based on a 33 gene expression signature. This could help select patients likely to respond to treatment with a primary radiotherapy approach as for anal cancer. Conversely, those with predicted radioresistance may be candidates for clinical trials evaluating addition of immune-oncology agents and stromal TGFβ signalling inhibition. FUNDING: The Stratification in Colorectal Cancer Consortium (S:CORT) was funded by the Medical Research Council and Cancer Research UK (MR/M016587/1).

New role of fat-free mass in cancer risk linked with genetic predisposition.

Cancer risk is associated with the widely debated measure body mass index (BMI). Fat mass and fat-free mass measurements from bioelectrical impedance may further clarify this association. The UK Biobank is a rare resource in which bioelectrical impedance and BMI data was collected on ~ 500,000 individuals. Using this dataset, a comprehensive analysis using regression, principal component and genome-wide genetic association, provided multiple levels of evidence that increasing whole body fat (WBFM) and fat-free mass (WBFFM) are both associated with increased post-menopausal breast cancer risk, and colorectal cancer risk in men. WBFM was inversely associated with prostate cancer. We also identified rs615029[T] and rs1485995[G] as associated in independent analyses with both PMBC (p = 1.56E-17 and 1.78E-11) and WBFFM (p = 2.88E-08 and 8.24E-12), highlighting splice variants of the intriguing long non-coding RNA CUPID1 (LINC01488) as a potential link between PMBC risk and fat-free mass.

Data from Targeting IGF Perturbs Global Replication through Ribonucleotide Reductase Dysfunction

<div>Abstract<p>Inhibition of IGF receptor (IGF1R) delays repair of radiation-induced DNA double-strand breaks (DSB), prompting us to investigate whether IGF1R influences endogenous DNA damage. Here we demonstrate that IGF1R inhibition generates endogenous DNA lesions protected by 53BP1 bodies, indicating under-replicated DNA. In cancer cells, inhibition or depletion of IGF1R delayed replication fork progression accompanied by activation of ATR–CHK1 signaling and the intra-S-phase checkpoint. This phenotype reflected unanticipated regulation of global replication by IGF1 mediated via AKT, MEK/ERK, and JUN to influence expression of ribonucleotide reductase (RNR) subunit RRM2. Consequently, inhibition or depletion of IGF1R downregulated RRM2, compromising RNR function and perturbing dNTP supply. The resulting delay in fork progression and hallmarks of replication stress were rescued by RRM2 overexpression, confirming RRM2 as the critical factor through which IGF1 regulates replication. Suspecting existence of a backup pathway protecting from toxic sequelae of replication stress, targeted compound screens in breast cancer cells identified synergy between IGF inhibition and ATM loss. Reciprocal screens of ATM-proficient/deficient fibroblasts identified an IGF1R inhibitor as the top hit. IGF inhibition selectively compromised growth of ATM-null cells and spheroids and caused regression of ATM-null xenografts. This synthetic-lethal effect reflected conversion of single-stranded lesions in IGF-inhibited cells into toxic DSBs upon ATM inhibition. Overall, these data implicate IGF1R in alleviating replication stress, and the reciprocal IGF:ATM codependence we identify provides an approach to exploit this effect in ATM-deficient cancers.</p>Significance:<p>This study identifies regulation of ribonucleotide reductase function and dNTP supply by IGFs and demonstrates that IGF axis blockade induces replication stress and reciprocal codependence on ATM.</p></div>

Structural basis for activity switching in polymerases determining the fate of let-7 pre-miRNAs.

Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3' end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A. Our structures reveal that pre-miRNAs engage the enzymes in fundamentally different ways depending on the presence of LIN28A, which clamps them onto the TUTs to enable processive 3' oligo-uridylation. This study reveals the molecular basis for mono- versus oligo-uridylation by TUT7/4, as determined by the presence of LIN28A, and thus their mechanism of action in the regulation of cell fate and in cancer.

Synthesis and Properties of Oligonucleotides Containing LNA-Sulfamate and Sulfamide Backbone Linkages.

Oligonucleotides hold great promise as therapeutic agents but poor bioavailability limits their utility. Hence, new analogues with improved cell uptake are urgently needed. Here, we report the synthesis and physical study of reduced-charge oligonucleotides containing artificial LNA-sulfamate and sulfamide linkages combined with 2'-O-methyl sugars and phosphorothioate backbones. These oligonucleotides have high affinity for RNA and excellent nuclease resistance.

Targeting PI3K-gamma in myeloid driven tumour immune suppression: a systematic review and meta-analysis of the preclinical literature.

The intricate interplay between immune and stromal cells within the tumour microenvironment (TME) significantly influences tumour progression. Myeloid cells, including tumour-associated macrophages (TAMs), neutrophils (TANs), and myeloid-derived suppressor cells (MDSCs), contribute to immune suppression in the TME (Nakamura and Smyth in Cell Mol Immunol 17(1):1-12 (2020). https://doi.org/10.1038/s41423-019-0306-1 ; DeNardo and Ruffell in Nat Rev Immunol 19(6):369-382 (2019). https://doi.org/10.1038/s41577-019-0127-6 ). This poses a significant challenge for novel immunotherapeutics that rely on host immunity to exert their effect. This systematic review explores the preclinical evidence surrounding the inhibition of phosphoinositide 3-kinase gamma (PI3Kγ) as a strategy to reverse myeloid-driven immune suppression in solid tumours. EMBASE, MEDLINE, and PubMed databases were searched on 6 October 2022 using keyword and subject heading terms to capture relevant studies. The studies, focusing on PI3Kγ inhibition in animal models, were subjected to predefined inclusion and exclusion criteria. Extracted data included tumour growth kinetics, survival endpoints, and immunological responses which were meta-analysed. PRISMA and MOOSE guidelines were followed. A total of 36 studies covering 73 animal models were included in the review and meta-analysis. Tumour models covered breast, colorectal, lung, skin, pancreas, brain, liver, prostate, head and neck, soft tissue, gastric, and oral cancer. The predominant PI3Kγ inhibitors were IPI-549 and TG100-115, demonstrating favourable specificity for the gamma isoform. Combination therapies, often involving chemotherapy, radiotherapy, immune checkpoint inhibitors, biological agents, or vaccines, were explored in 81% of studies. Analysis of tumour growth kinetics revealed a statistically significant though heterogeneous response to PI3Kγ monotherapy, whereas the tumour growth in combination treated groups were more consistently reduced. Survival analysis showed a pronounced increase in median overall survival with combination therapy. This systematic review provides a comprehensive analysis of preclinical studies investigating PI3Kγ inhibition in myeloid-driven tumour immune suppression. The identified studies underscore the potential of PI3Kγ inhibition in reshaping the TME by modulating myeloid cell functions. The combination of PI3Kγ inhibition with other therapeutic modalities demonstrated enhanced antitumour effects, suggesting a synergistic approach to overcome immune suppression. These findings support the potential of PI3Kγ-targeted therapies, particularly in combination regimens, as a promising avenue for future clinical exploration in diverse solid tumour types.