KRAS4A induces metastatic lung adenocarcinomas in vivo in the absence of the KRAS4B isoform.

In mammals, the KRAS locus encodes two protein isoforms, KRAS4A and KRAS4B, which differ only in their C terminus via alternative splicing of distinct fourth exons. Previous studies have shown that whereas KRAS expression is essential for mouse development, the KRAS4A isoform is expendable. Here, we have generated a mouse strain that carries a terminator codon in exon 4B that leads to the expression of an unstable KRAS4B154 truncated polypeptide, hence resulting in a bona fide Kras4B-null allele. In contrast, this terminator codon leaves expression of the KRAS4A isoform unaffected. Mice selectively lacking KRAS4B expression developed to term but died perinatally because of hypertrabeculation of the ventricular wall, a defect reminiscent of that observed in embryos lacking the Kras locus. Mouse embryonic fibroblasts (MEFs) obtained from Kras4B-/- embryos proliferated less than did wild-type MEFs, because of limited expression of KRAS4A, a defect that can be compensated for by ectopic expression of this isoform. Introduction of the same terminator codon into a KrasFSFG12V allele allowed expression of an endogenous KRAS4AG12V oncogenic isoform in the absence of KRAS4B. Exposure of Kras+/FSF4AG12V4B- mice to Adeno-FLPo particles induced lung tumors with complete penetrance, albeit with increased latencies as compared with control Kras+/FSFG12V animals. Moreover, a significant percentage of these mice developed proximal metastasis, a feature seldom observed in mice expressing both mutant isoforms. These results illustrate that expression of the KRAS4AG12V mutant isoform is sufficient to induce lung tumors, thus suggesting that selective targeting of the KRAS4BG12V oncoprotein may not have significant therapeutic consequences.

miR-203 drives breast cancer cell differentiation.

A hallmark of many malignant tumors is dedifferentiated (immature) cells bearing slight or no resemblance to the normal cells from which the cancer originated. Tumor dedifferentiated cells exhibit a higher capacity to survive to chemo and radiotherapies and have the ability to incite tumor relapse. Inducing cancer cell differentiation would abolish their self-renewal and invasive capacity and could be combined with the current standard of care, especially in poorly differentiated and aggressive tumors (with worst prognosis). However, differentiation therapy is still in its early stages and the intrinsic complexity of solid tumor heterogeneity demands innovative approaches in order to be efficiently translated into the clinic. We demonstrate here that microRNA 203, a potent driver of differentiation in pluripotent stem cells (ESCs and iPSCs), promotes the differentiation of mammary gland tumor cells. Combining mouse in vivo approaches and both mouse and human-derived tridimensional organoid cultures, we report that miR-203 influences the self-renewal capacity, plasticity and differentiation potential of breast cancer cells and prevents tumor cell growth in vivo. Our work sheds light on differentiation-based antitumor therapies and offers miR-203 as a promising tool for directly confronting the tumor-maintaining and regeneration capability of cancer cells.

Tumor regression and resistance mechanisms upon CDK4 and RAF1 inactivation in KRAS/P53 mutant lung adenocarcinomas.

KRAS mutant lung adenocarcinomas remain intractable for targeted therapies. Genetic interrogation of KRAS downstream effectors, including the MAPK pathway and the interphase CDKs, identified CDK4 and RAF1 as the only targets whose genetic inactivation induces therapeutic responses without causing unacceptable toxicities. Concomitant CDK4 inactivation and RAF1 ablation prevented tumor progression and induced complete regression in 25% of KRAS/p53-driven advanced lung tumors, yet a significant percentage of those tumors that underwent partial regression retained a population of CDK4/RAF1-resistant cells. Characterization of these cells revealed two independent resistance mechanisms implicating hypermethylation of several tumor suppressors and increased PI3K activity. Importantly, these CDK4/RAF1-resistant cells can be pharmacologically controlled. These studies open the door to new therapeutic strategies to treat KRAS mutant lung cancer, including resistant tumors.

FGFR1 amplification or overexpression and hormonal resistance in luminal breast cancer: rationale for a triple blockade of ER, CDK4/6, and FGFR1.

BACKGROUND: FGFR1 amplification, but not overexpression, has been related to adverse prognosis in hormone-positive breast cancer (HRPBC). Whether FGFR1 overexpression and amplification are correlated, what is their distribution among luminal A or B HRPBC, and if there is a potential different prognostic role for amplification and overexpression are currently unknown features. The role of FGFR1 inhibitors in HRPBC is also unclear. METHODS: FGFR1 amplification (FISH) and overexpression (RNAscope) were investigated in a N = 251 HRPBC patients cohort and the METABRIC cohort; effects on survival and FISH-RNAscope concordance were determined. We generated hormonal deprivation resistant (LTED-R) and FGFR1-overexpressing cell line variants of the ER+ MCF7 and T47-D and the ER+, FGFR1-amplified HCC1428 cell lines. The role of ER, CDK4/6, and/or FGFR1 blockade alone or in combinations in Rb phosphorylation, cell cycle, and survival were studied. RESULTS: FGFR1 overexpression and amplification was non-concordant in > 20% of the patients, but both were associated to a similar relapse risk (~ 2.5-fold; P < 0.05). FGFR1 amplification or overexpression occurred regardless of the luminal subtype, but the incidence was higher in luminal B (16.3%) than A (6.6%) tumors; P < 0.05. The Kappa index for overexpression and amplification was 0.69 (P < 0.001). Twenty-four per cent of the patients showed either amplification and/or overexpression of FGFR1, what was associated to a hazard ratio for relapse of 2.6 (95% CI 1.44-4.62, P < 0.001). In vitro, hormonal deprivation led to FGFR1 overexpression. Primary FGFR1 amplification, engineered mRNA overexpression, or LTED-R-acquired FGFR1 overexpression led to resistance against hormonotherapy alone or in combination with the CDK4/6 inhibitor palbociclib. Blocking FGFR1 with the kinase-inhibitor rogaratinib led to suppression of Rb phosphorylation, abrogation of the cell cycle, and resistance-reversion in all FGFR1 models. CONCLUSIONS: FGFR1 amplification and overexpression are associated to similar adverse prognosis in hormone-positive breast cancer. Capturing all the patients with adverse prognosis-linked FGFR1 aberrations requires assessing both features. Hormonal deprivation leads to FGFR1 overexpression, and FGFR1 overexpression and/or amplification are associated with resistance to hormonal monotherapy or in combination with palbociclib. Both resistances are reverted with triple ER, CDK4/6, and FGFR1 blockade.

Prevalence of pathogenic germline variants in patients with metastatic renal cell carcinoma.

PURPOSE: Germline pathogenic variants are estimated to affect 3-5% of renal cell carcinoma (RCC) patients. However, higher mutational prevalence in non-clear cell RCC (non-ccRCC) and advanced disease has been suggested. METHODS: To clarify the prevalence of pathogenic germline variants in metastatic RCC, we sequenced 29 cancer susceptibility genes in 294 unselected metastatic RCC cases plus 21 patients with clinical hereditary features. In 145 tumors, genes frequently mutated in RCC were sequenced and methylation was assessed in selected cases. RESULTS: Germline variants in RCC predisposition genes (FH, VHL) were detected in 1.4% of the unselected metastatic patients, with higher frequency in non-ccRCC versus ccRCC (6.4% and 0.4%; P = 0.0025) and in younger patients (P = 0.036). Among the 315 studied patients, 14% of non-type 1 papillary cases (4 of 28), all metastatic <1 year after diagnosis, carried a FH germline variant with loss of heterozygosity and tumor genome hypermethylation. Variants in other cancer-associated genes (e.g., MUTYH, BRCA2, CHEK2) occurred in 5.1% of the unselected series, with unclear significance for RCC. CONCLUSION: Our findings confirm a high prevalence of pathogenic germline variants in RCC predisposition genes in metastatic non-ccRCC, and highlight that metastatic patients with papillary type 2 or unconventional histologies compatible with FH would benefit from genetic screening.

Metabolomics, machine learning and immunohistochemistry to predict succinate dehydrogenase mutational status in phaeochromocytomas and paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background in over one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and several other tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlighting the importance of identifying SDHx mutations for patient management. Genetic variants of unknown significance, where implications for the patient and family members are unclear, are a problem for interpretation. For such cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB (SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatography-mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions provide an alternative method. Here, we compare SDHB-IHC with metabolite profiling in 189 tumours from 187 PPGL patients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to establish predictive models for interpreting metabolite data. Metabolite profiling showed higher diagnostic specificity compared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite profiles improved predictive ability over that of the SFR, in particular for hard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, the combination of metabolite profiling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classified all but one of the false negatives from metabolite profiling strategies, while metabolite profiling correctly classified all but one of the false negatives/positives from SDHB-IHC. From 186 tumours with confirmed status of SDHx variant pathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benefits of both strategies for patient management. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Immuno-priming durvalumab with bevacizumab in HER2-negative advanced breast cancer: a pilot clinical trial.

BACKGROUND: Preclinical research suggests that the efficacy of immune checkpoint inhibitors in breast cancer can be enhanced by combining them with antiangiogenics, particularly in a sequential fashion. We sought to explore the efficacy and biomarkers of combining the anti-PD-L1 durvalumab plus the antiangiogenic bevacizumab after bevacizumab monotherapy for advanced HER2-negative breast cancer. METHODS: Patients had advanced HER2-negative disease that progressed while receiving single-agent bevacizumab maintenance as a part of a previous chemotherapy plus bevacizumab regimen. Treatment consisted of bi-weekly durvalumab plus bevacizumab (10 mg/kg each i.v.). Peripheral-blood mononuclear cells (PBMCs) were obtained before the first durvalumab dose and every 4 weeks and immunophenotyped by flow-cytometry. A fresh pre-durvalumab tumor biopsy was obtained; gene-expression studies and immunohistochemical staining to assess vascular normalization and characterize the immune infiltrate were conducted. Patients were classified as "non-progressors" if they had clinical benefit (SD/PR/CR) at 4 months. The co-primary endpoints were the changes in the percentage T cell subpopulations in PBMCs in progressors versus non-progressors, and PFS/OS time. RESULTS: Twenty-six patients were accrued. Median PFS and OS were 3.5 and 11 months; a trend for a longer OS was detected for the hormone-positive subset (19.8 versus 7.4 months in triple-negatives; P = 0.11). Clinical benefit rate at 2 and 4 months was 60% and 44%, respectively, without significant differences between hormone-positive and triple-negative (P = 0.73). Non-progressors' tumors displayed vascular normalization features as a result of previous bevacizumab, compared with generally abnormal patterns observed in progressors. Non-progressors also showed increased T-effector and T-memory signatures and decreased TREG signatures in gene expression studies in baseline-post-bevacizumab-tumors compared with progressors. Notably, analysis of PBMC populations before durvalumab treatment was concordant with the findings in tumor samples and showed a decreased percentage of circulating TREGs in non-progressors. CONCLUSIONS: This study reporting on sequential bevacizumab+durvalumab in breast cancer showed encouraging activity in a heavily pre-treated cohort. The correlative studies agree with the preclinical rationale supporting an immunopriming effect exerted by antiangiogenic treatment, probably by reducing TREGs cells both systemically and in tumor tissue. The magnitude of this benefit should be addressed in a randomized setting. TRIAL REGISTRATION: (www.clinicaltrials.gov): NCT02802098 . Registered on June 16, 2020.

PTEN expression and mutations in TSC1, TSC2 and MTOR are associated with response to rapalogs in patients with renal cell carcinoma.

The mammalian target of rapamycin (mTOR) pathway inhibitors are key drugs for the treatment of many tumor types, however, there are no predictive biomarkers in clinical use. Here, we performed a molecular and immunohistochemical characterization of key mTOR pathway components in a series of 105 renal cell carcinoma patients treated with rapalogs, aimed at identifying markers of treatment response. Mutational analysis in MTOR, TSC1 and TSC2 was performed through targeted next-generation sequencing (NGS), and immunohistochemistry (IHC) was performed for PTEN, pAKT, pS6K1, pS6 and p21. Among patients with NGS data, 11 of 87 (13%) had mTOR pathway mutations (8 in MTOR, 1 in TSC1 and 2 in TSC2). When comparing the molecular data to the response of the patients, we found that partial response was more frequent in cases with mTOR pathway mutations than in those without mutations (odds ratio [OR] = 0.08, 95% confidence interval [CI] = 0.008-0.79, p = 0.030 univariate; p = 0.038 multivariable). Regarding IHC, negative PTEN staining was detected in 58% of the tumors, and it was more frequent in rapalog responder patients (OR = 0.24, 95% CI = 0.065-0.86, p = 0.029 univariate; p = 0.029 multivariable). Mutations and PTEN IHC were not mutually exclusive events and its combination improved response prediction (OR = 0.16, 95% CI = 0.04-0.62, p = 0.008 univariate; p = 0.013 multivariable). The staining of other proteins did not show and association with response and no association with PFS was observed in unselected patients. In conclusion, our findings suggest that mTOR pathway mutations, negative PTEN IHC and their combination are potential markers of rapalog response.

Molecular characterization of chromophobe renal cell carcinoma reveals mTOR pathway alterations in patients with poor outcome.

Chromophobe renal cell carcinoma (chRCC) is a histologically and molecularly distinct class of rare renal tumor. TCGA studies revealed low mutational burden, with only TP53 and PTEN recurrently mutated, and discovered alterations in TERT promoter and in the electron transport chain Complex I genes. However, knowledge on drug targetable genes is limited and treatments at metastatic stage do not follow a molecular rationale. In a large series of 92 chRCC enriched with metastatic cases, we performed an in-depth characterization of mTOR pathway alterations through targeted NGS and immunohistochemistry (IHC) of phospho-S6, tuberin, and PTEN. Mutations in mitochondria, telomere maintenance and other renal cancer related genes and p53 IHC, were also assessed. The impact on metastasis development and disease specific survival was determined, using TCGA-KICH series (n = 65) for validation. mTOR pathway mutations (MTOR, TSC1, TSC2) were present in 17% of primary tumors, most of them being classified as pathogenic. Mutations were associated with positive IHC staining of phospho-S6 and PTEN (P = 0.009 and P = 0.001, respectively) and with chRCC eosinophilic variant (P = 0.039), supporting a biological relevance of the pathway. mTOR pathway mutations were associated with worse clinical outcomes. Survival analysis gave a hazard ratio of 5.5 (P = 0.027), and this association was confirmed in TCGA-KICH (HR = 10.3, P = 0.006). TP53 mutations were enriched in metastatic cases (P = 0.018), and mutations in telomere maintenance genes showed a trend in the same direction. p53 IHC staining pattern was associated with the underlying TP53 defect, and negative PTEN IHC staining (82% of cases) suggested PTEN loss as a chRCC hallmark. In conclusion, our study provides with novel genomic knowledge in chRCC and identifies novel markers of poor survival. Furthermore, this is the first study showing that mTOR pathway mutations correlate with poor prognosis, and may help to identify patients with increased sensitivity to mTOR inhibitors.

Subclassification of the molecular types of breast cancer based on the expression of immunohistochemical markers and evolution.

Breast carcinomas have been classified from the molecular point of view into four major groups (Luminal A, Luminal B, HER2 and triple negative). However, these groups are not homogeneous and there is a need to establish subcategories that can be identified by immunohistochemistry (IHC), to better predict prognosis and carry out treatments that are more effective. This study was conducted in 354 patients diagnosed with invasive ductal breast carci- noma. The expression of 22 molecules was analyzed by tissue matrices and the results obtained were compared with molecular classes defined by IHC, according to the expression of estrogen receptor (ER), progesterone receptor (PR) and HER2, and the overall survival. The Luminal A molecular class can set various prognosticosubgroups: the subgroup with exclusive expression of ER and PR, with Ki-67 ≤14%, with a better prognosis, and the subgroup of Luminal A with Ki-67> 14% or other expression related to the basal phenotype markers. Luminal B group can be divided into subtypes according to the expression of Ki-67 (cutoff at 25%). In the HER2 class it seems important the Ki-67 index for forecasting (cutoff at 25%). The TN class can be divided according to the rate of proliferation into two prognostic categories, with a better prog- nosis for tumors with Ki-67 index ≤25%.

[Cadherins E and P expression in the molecular types of breast cancer]. / Expresión de cadherinas E y P en los tipos moleculares de cáncer de mama.

The epithelial-mesenchymal transition is a process by which tumor cells lose their epithelial markers and migrate to distant organs. This process involves several cell adhesion proteins such as E-cadherin and P-cadherin. The present study was performed in 354 pacients diagnosed with breast infiltrating ductal carcinoma in the Oncology Institute "Dr. Miguel Perez Carreno", Valencia, Venezuela. The expression of 22 molecules was analyzed by tissue micro-arrays and the results were compared with the molecular clases established by immunohistochemistry, according to the'expression of estrogen receptor (ER), progesterone (PR) and human epidermal growth factor receptor type 2 (HER2), and with the overall survival (OS). Based on the results of ER, PR and HER2 molecular classes according to the following per- centages were established: Luminal A 42.4%, Luminal B 20.3%, 9% HER2 and 28.2% triple negative (TN). E-cadherin expression was observed conserved in most of the tumors of this series, 92.5% of cases. TN phenotype tumors showed a high percentage (41.7%) with absent or reduced expression. The P-cadherin was expressed in 40.5% of cases, although expressed in all classes; the proportion was significantly higher in cases TN. No significant prognostic value was observed when analyzing the overall five-year survival of patients with tumors with absent or reduced expression of E-cadherin. The P-cadherin expression had a negative relationship with the OS.

Distribution of PD-L1, TROP2 and HER2- "lowness" in early triple-negative breast cancer: an opportunity for treatment de-escalation.

BACKGROUND: HER2, TROP2 and PD-L1 are novel targets in triple-negative breast cancer (TNBC). The combined expression status of these targets, and whether they can define prognostic subgroups, is currently undefined. METHODS: Immunohistochemistry was used to determine HER2, TROP2 and PD-L1 levels in 459 TNBC cases, that received in the adjuvant/neoadjuvant setting active surveillance, CMF, anthracycline-, anthracycline plus taxane-, or carboplatin-containing regimes. RESULTS: HER2-low patients with PD-L1 > 1 CPS (double-positive, herein "DP") had a mean PFS of 4768 days (95% CI: 4267-5268) versus 3522 days (95% CI: 3184-3861) for non-DP patients (P = 0.002). Regarding the received adjuvant treatment, DP patients (versus non-DP) receiving anthracyclines plus taxanes exhibited a mean PFS time of 4726 (95% CI: 4022-5430) versus 3302 (95% CI: 2818-3785) days (P = 0.039). Finally, 100% of DP patients that received a carboplatin-based regimen were long-term disease-free. CONCLUSIONS: Early HER2-low, PD-L1-positive TNBC patients have a very good prognosis, particularly if treated with anthracycline/taxane- or carboplatin-containing regimes.

NGFR regulates stromal cell activation in germinal centers.

Nerve growth factor receptor (NGFR) is expressed by follicular dendritic cells (FDCs). However, the role of NGFR in the humoral response is not well defined. Here, we study the effect of Ngfr loss on lymph node organization and function, demonstrating that Ngfr depletion leads to spontaneous germinal center (GC) formation and an expansion of the GC B cell compartment. In accordance with this effect, stromal cells are altered in Ngfr-/- mice with a higher frequency of FDCs, characterized by CD21/35, MAdCAM-1, and VCAM-1 overexpression. GCs are located ectopically in Ngfr-/- mice, with lost polarization together with impaired high-affinity antibody production and an increase in circulating autoantibodies. We observe higher levels of autoantibodies in Bcl2 Tg/Ngfr-/- mice, concomitant with a higher incidence of autoimmunity and lower overall survival. Our work shows that NGFR is involved in maintaining GC structure and function, participating in GC activation, antibody production, and immune tolerance.

Hepatic nutrient and hormone signaling to mTORC1 instructs the postnatal metabolic zonation of the liver.

The metabolic functions of the liver are spatially organized in a phenomenon called zonation, linked to the differential exposure of portal and central hepatocytes to nutrient-rich blood. The mTORC1 signaling pathway controls cellular metabolism in response to nutrients and insulin fluctuations. Here we show that simultaneous genetic activation of nutrient and hormone signaling to mTORC1 in hepatocytes results in impaired establishment of postnatal metabolic and zonal identity of hepatocytes. Mutant hepatocytes fail to upregulate postnatally the expression of Frizzled receptors 1 and 8, and show reduced Wnt/ß-catenin activation. This defect, alongside diminished paracrine Wnt2 ligand expression by endothelial cells, underlies impaired postnatal maturation. Impaired zonation is recapitulated in a model of constant supply of nutrients by parenteral nutrition to piglets. Our work shows the role of hepatocyte sensing of fluctuations in nutrients and hormones for triggering a latent metabolic zonation program.

A mild increase in nutrient signaling to mTORC1 in mice leads to parenchymal damage, myeloid inflammation and shortened lifespan.

The mechanistic target of rapamycin complex 1 controls cellular anabolism in response to growth factor signaling and to nutrient sufficiency signaled through the Rag GTPases. Inhibition of mTOR reproducibly extends longevity across eukaryotes. Here we report that mice that endogenously express active mutant variants of RagC exhibit multiple features of parenchymal damage that include senescence, expression of inflammatory molecules, increased myeloid inflammation with extensive features of inflammaging and a ~30% reduction in lifespan. Through bone marrow transplantation experiments, we show that myeloid cells are abnormally activated by signals emanating from dysfunctional RagC-mutant parenchyma, causing neutrophil extravasation that inflicts additional inflammatory damage. Therapeutic suppression of myeloid inflammation in aged RagC-mutant mice attenuates parenchymal damage and extends survival. Together, our findings link mildly increased nutrient signaling to limited lifespan in mammals, and support a two-component process of parenchymal damage and myeloid inflammation that together precipitate a time-dependent organ deterioration that limits longevity.

Cabergoline as a Novel Strategy for Post-Pregnancy Breast Cancer Prevention in Mice and Human.

Post-pregnancy breast cancer often carries a poor prognosis, posing a major clinical challenge. The increasing trend of later-life pregnancies exacerbates this risk, highlighting the need for effective chemoprevention strategies. Current options, limited to selective estrogen receptor modulators, aromatase inhibitors, or surgical procedures, offer limited efficacy and considerable side effects. Here, we report that cabergoline, a dopaminergic agonist, reduces the risk of breast cancer post-pregnancy in a Brca1/P53-deficient mouse model, with implications for human breast cancer prevention. We show that a single dose of cabergoline administered post-pregnancy significantly delayed the onset and reduced the incidence of breast cancer in Brca1/P53-deficient mice. Histological analysis revealed a notable acceleration in post-lactational involution over the short term, characterized by increased apoptosis and altered gene expression related to ion transport. Over the long term, histological changes in the mammary gland included a reduction in the ductal component, decreased epithelial proliferation, and a lower presence of recombinant Brca1/P53 target cells, which are precursors of tumors. These changes serve as indicators of reduced breast cancer susceptibility. Additionally, RNA sequencing identified gene expression alterations associated with decreased proliferation and mammary gland branching. Our findings highlight a mechanism wherein cabergoline enhances the protective effect of pregnancy against breast cancer by potentiating postlactational involution. Notably, a retrospective cohort study in women demonstrated a markedly lower incidence of post-pregnancy breast cancer in those treated with cabergoline compared to a control group. Our work underscores the importance of enhancing postlactational involution as a strategy for breast cancer prevention, and identifies cabergoline as a promising, low-risk option in breast cancer chemoprevention. This strategy has the potential to revolutionize breast cancer prevention approaches, particularly for women at increased risk due to genetic factors or delayed childbirth, and has wider implications beyond hereditary breast cancer cases.

Genetic deletion of HVEM in a leukemia B cell line promotes a preferential increase of PD-1- stem cell-like T cells over PD-1+ T cells curbing tumor progression.

Introduction: A high frequency of mutations affecting the gene encoding Herpes Virus Entry Mediator (HVEM, TNFRSF14) is a common clinical finding in a wide variety of human tumors, including those of hematological origin. Methods: We have addressed how HVEM expression on A20 leukemia cells influences tumor survival and its involvement in the modulation of the anti-tumor immune responses in a parental into F1 mouse tumor model of hybrid resistance by knocking-out HVEM expression. HVEM WT or HVEM KO leukemia cells were then injected intravenously into semiallogeneic F1 recipients and the extent of tumor dissemination was evaluated. Results: The loss of HVEM expression on A20 leukemia cells led to a significant increase of lymphoid and myeloid tumor cell infiltration curbing tumor progression. NK cells and to a lesser extent NKT cells and monocytes were the predominant innate populations contributing to the global increase of immune infiltrates in HVEM KO tumors compared to that present in HVEM KO tumors. In the overall increase of the adaptive T cell immune infiltrates, the stem cell-like PD-1- T cells progenitors and the effector T cell populations derived from them were more prominently present than terminally differentiated PD-1+ T cells. Conclusions: These results suggest that the PD-1- T cell subpopulation is likely to be a more relevant contributor to tumor rejection than the PD-1+ T cell subpopulation. These findings highlight the role of co-inhibitory signals delivered by HVEM upon engagement of BTLA on T cells and NK cells, placing HVEM/BTLA interaction in the spotlight as a novel immune checkpoint for the reinforcement of the anti-tumor responses in malignancies of hematopoietic origin.

PBRM1 and KDM5C cooperate to define high-angiogenesis tumors and increased antiangiogenic response in renal cancer.

Vascular endothelial growth factor receptor tyrosine kinase inhibitors (VEGFR-TKIs) are key antiangiogenic drugs for renal cancer treatment. While Von Hippel-Lindau dysfunction constitutes the base for VEGFR-TKIs sensitivity, the role for individual and concurrent mutations in the genes encoding for the chromatin remodelers Polybromo-1 (PBRM1) and Lysine Demethylase 5C (KDM5C) is poorly understood. Here, we analyzed the tumor mutational and expression profiles of 155 unselected clear cell RCC (ccRCC) cases treated with first-line VEGFR-TKIs and the ccRCC cases of IMmotion151 trial were used for validation. We found that concurrent PBRM1 and KDM5C (PBRM1&KDM5C) mutations occurred in 4-9% of cases and were enriched in Memorial Sloan Kettering Cancer Center favorable-risk patients. In our cohort, tumors only mutated in PBRM1 or concurrently mutated in PBRM1 and KDM5C had increased angiogenesis (P=0.0068 and 0.039; respectively), and tumors only mutated in KDM5C showed a similar trend. Best response to VEGFR-TKIs corresponded to PBRM1&KDM5C mutated cases, followed by those mutated only in KDM5C or only in PBRM1 (P=0.050, 0.040 and 0.027 versus non-mutated cases, respectively), with a trend for longer progression free survival (PFS) in the group with only PBRM1 mutated (HR=0.64; P=0.059). Validation in the IMmotion151 trial revealed a similar correlation with increased angiogenesis and the PFS of patients in the VEGFR-TKI-arm was the longest in PBRM1&KDM5C mutated cases, intermediate for only PBRM1 or only KDM5C mutated patients and the shortest in non-mutated cases (P=0.009 and 0.025, for PBRM1&KDM5C and PBRM1 versus non-mutated cases). In conclusion, somatic PBRM1 and KDM5C mutations are common in patients with metastatic ccRCC and likely cooperate increasing tumor angiogenesis and VEGFR-TKI-based antiangiogenic therapy benefit.

Mitochondrial RNA methyltransferase TRMT61B is a new, potential biomarker and therapeutic target for highly aneuploid cancers.

Despite being frequently observed in cancer cells, chromosomal instability (CIN) and its immediate consequence, aneuploidy, trigger adverse effects on cellular homeostasis that need to be overcome by anti-stress mechanisms. As such, these safeguard responses represent a tumor-specific Achilles heel, since CIN and aneuploidy are rarely observed in normal cells. Recent data have revealed that epitranscriptomic marks catalyzed by RNA-modifying enzymes change under various stress insults. However, whether aneuploidy is associated with such RNA modifying pathways remains to be determined. Through an in silico search for aneuploidy biomarkers in cancer cells, we found TRMT61B, a mitochondrial RNA methyltransferase enzyme, to be associated with high levels of aneuploidy. Accordingly, TRMT61B protein levels are increased in tumor cell lines with an imbalanced karyotype as well as in different tumor types when compared to control tissues. Interestingly, while TRMT61B depletion induces senescence in melanoma cell lines with low levels of aneuploidy, it leads to apoptosis in cells with high levels. The therapeutic potential of these results was further validated by targeting TRMT61B in transwell and xenografts assays. We show that TRM61B depletion reduces the expression of several mitochondrial encoded proteins and limits mitochondrial function. Taken together, these results identify a new biomarker of aneuploidy in cancer cells that could potentially be used to selectively target highly aneuploid tumors.

Kras oncogene ablation prevents resistance in advanced lung adenocarcinomas.

KRASG12C inhibitors have revolutionized the clinical management of patients with KRASG12C-mutant lung adenocarcinoma. However, patient exposure to these inhibitors leads to the rapid onset of resistance. In this study, we have used genetically engineered mice to compare the therapeutic efficacy and the emergence of tumor resistance between genetic ablation of mutant Kras expression and pharmacological inhibition of oncogenic KRAS activity. Whereas Kras ablation induces massive tumor regression and prevents the appearance of resistant cells in vivo, treatment of KrasG12C/Trp53-driven lung adenocarcinomas with sotorasib, a selective KRASG12C inhibitor, caused a limited antitumor response similar to that observed in the clinic, including the rapid onset of resistance. Unlike in human tumors, we did not observe mutations in components of the RAS-signaling pathways. Instead, sotorasib-resistant tumors displayed amplification of the mutant Kras allele and activation of xenobiotic metabolism pathways, suggesting that reduction of the on-target activity of KRASG12C inhibitors is the main mechanism responsible for the onset of resistance. In sum, our results suggest that resistance to KRAS inhibitors could be prevented by achieving a more robust inhibition of KRAS signaling mimicking the results obtained upon Kras ablation.