The Spindle Assembly Checkpoint Regulates the Phosphorylation State of a Subset of DNA Checkpoint Proteins in Saccharomyces cerevisiae

The DNA and the spindle assembly checkpoints play key roles in maintaining genomic integrity by coordinating cell responses to DNA lesions and spindle dysfunctions, respectively. These two surveillance pathways seem to operate mostly independently of one another, and little is known about their potential physiological connections. Here, we show that in Saccharomyces cerevisiae, the activation of the spindle assembly checkpoint triggers phosphorylation changes in two components of the DNA checkpoint, Rad53 and Rad9. These modifications are independent of the other DNA checkpoint proteins and are abolished in spindle checkpoint-defective mutants, hinting at specific functions for Rad53 and Rad9 in the spindle damage response. Moreover, we found that after UV irradiation, Rad9 phosphorylation is altered and Rad53 inactivation is accelerated when the spindle checkpoint is activated, which suggests the implication of the spindle checkpoint in the regulation of the DNA damage response

Optimal dosing regimen of CD73 blockade improves tumor response to radiotherapy through iCOS downregulation

Background Irradiation (IR) and immune checkpoint inhibitor (ICI) combination is a promising treatment modality. However, local and distance treatment failure and resistance can occur. To counteract this resistance, several studies propose CD73, an ectoenzyme, as a potential target to improve the antitumor efficiency of IR and ICI. Although CD73 targeting in combination with IR and ICI has shown attractive antitumor effects in preclinical models, the rationale for CD73 targeting based on CD73 tumor expression level deserves further investigations.Methods Here we evaluated for the first time the efficacy of two administration regimens of CD73 neutralizing antibody (one dose vs four doses) in combination with IR according to the expression level of CD73 in two subcutaneous tumor models expressing different levels of CD73.Results We showed that CD73 is weakly expressed by MC38 tumors even after IR, when compared with the TS/A model that highly expressed CD73. Treatment with four doses of anti-CD73 improved the TS/A tumor response to IR, while it was ineffective against the CD73 low-expressing MC38 tumors. Surprisingly, a single dose of anti-CD73 exerted a significant antitumor activity against MC38 tumors. On CD73 overexpression in MC38 cells, four doses of anti-CD73 were required to improve the efficacy of IR. Mechanistically, a correlation between a downregulation of iCOS expression in CD4+ T cells and an improved response to IR after anti-CD73 treatment was observed and iCOS targeting could restore an impaired benefit from anti-CD73 treatment.Conclusions These data emphasize the importance of the dosing regimen for anti-CD73 treatment to improve tumor response to IR and identify iCOS as part of the underlying molecular mechanisms. Our data suggest that the selection of appropriate dosing regimen is required to optimize the therapeutic efficacy of immunotherapy–radiotherapy combinations

TGFβ receptor inhibition unleashes interferon-β production by tumor-associated macrophages and enhances radiotherapy efficacy

International audienceBackground Transforming growth factor-beta (TGFβ) can limit the efficacy of cancer treatments, including radiotherapy (RT), by inducing an immunosuppressive tumor environment. The association of TGFβ with impaired T cell infiltration and antitumor immunity is known, but the mechanisms by which TGFβ participates in immune cell exclusion and limits the efficacy of antitumor therapies warrant further investigations. Methods We used the clinically relevant TGFβ receptor 2 (TGFβR2)-neutralizing antibody MT1 and the small molecule TGFβR1 inhibitor LY3200882 and evaluated their efficacy in combination with RT against murine orthotopic models of head and neck and lung cancer. Results We demonstrated that TGFβ pathway inhibition strongly increased the efficacy of RT. TGFβR2 antibody upregulated interferon beta expression in tumor-associated macrophages within the irradiated tumors and favored T cell infiltration at the periphery and within the core of the tumor lesions. We highlighted that both the antitumor efficacy and the increased lymphocyte infiltration observed with the combination of MT1 and RT were dependent on type I interferon signaling. Conclusions These data shed new light on the role of TGFβ in limiting the efficacy of RT, identifying a novel mechanism involving the inhibition of macrophage-derived type I interferon production, and fostering the use of TGFβR inhibition in combination with RT in therapeutic strategies for the management of head and neck and lung cancer

KRASG12C inhibition using MRTX1257: a novel radio-sensitizing partner

Abstract Background KRAS activating mutations are considered the most frequent oncogenic drivers and are correlated with radio-resistance in multiple cancers including non-small cell lung cancer (NSCLC) and colorectal cancer. Although KRAS was considered undruggable until recently, several KRAS inhibitors have recently reached clinical development. Among them, MRTX849 (Mirati Therapeutics) showed encouraging clinical outcomes for the treatment of selected patients with KRAS G12C mutated NSCLC and colorectal cancers. In this work, we explore the ability of MRTX1257, a KRASG12C inhibitor analogous to MRTX849, to radio-sensitize KRAS G12C+/+ mutated cell lines and tumors. Methods Both in vitro and in vivo models of radiotherapy (RT) in association with MRTX1257 were used, with different RAS mutational profiles. We assessed in vitro the radio-sensitizing effect of MRTX1257 in CT26 KRASG12C+/+, CT26 WT, LL2 WT and LL2 NRAS KO (LL2 NRAS−/−) cell lines. In vivo, we used syngeneic models of subcutaneous CT26 KRASG12C+/+ tumors in BALB/c mice and T cell deficient athymic nu/nu mice to assess both the radio-sensitizing effect of MRTX1257 and its immunological features. Results MRTX1257 was able to radio-sensitize CT26 KRASG12C+/+ cells in vitro in a time and dose dependent manner. Moreover, RT in association with MRTX1257 in BALB/c mice bearing CT26 KRASG12C+/+ subcutaneous tumors resulted in an observable cure rate of 20%. However, no durable response was observed with similar treatment in athymic nude mice. The analysis of the immune microenvironment of CT26 KRASG12C+/+ tumors following RT and MRTX1257 showed an increase in the proportion of various cell subtypes including conventional CD4 + T cells, dendritic cells type 2 (cDC2) and inflammatory monocytes. Furthermore, the expression of PD-L1 was dramatically down-regulated within both tumor and myeloid cells, thus illustrating the polarization of the tumor microenvironment towards a pro-inflammatory and anti-tumor phenotype following the combined treatment. Conclusion This work is the first to demonstrate in vitro as in vivo the radio-sensitizing effect of MRTX1257, a potent KRASG12C inhibitor compatible with oral administration, in CT26 KRASG12C mutated cell lines and tumors. This is a first step towards the use of new combinatorial strategies using KRAS inhibitors and RT in KRASG12C mutated tumors, which are the most represented in NSCLC with 14% of patients harboring this mutational profile

LymphoDose: a lymphocyte dose estimation framework—application to brain radiotherapy

International audienceAbstract Objective . Severe radiation-induced lymphopenia occurs in 40% of patients treated for primary brain tumors and is an independent risk factor of poor survival outcomes. We developed an in-silico framework that estimates the radiation doses received by lymphocytes during volumetric modulated arc therapy brain irradiation. Approach . We implemented a simulation consisting of two interconnected compartmental models describing the slow recirculation of lymphocytes between lymphoid organs ( M 1 ) and the bloodstream ( M 2 ). We used dosimetry data from 33 patients treated with chemo-radiation for glioblastoma to compare three cases of the model, corresponding to different physical and biological scenarios: (H1) lymphocytes circulation only in the bloodstream i.e. circulation in M 2 only; (H2) lymphocytes recirculation between lymphoid organs i.e. circulation in M 1 and M 2 interconnected; (H3) lymphocytes recirculation between lymphoid organs and deep-learning computed out-of-field (OOF) dose to head and neck (H&N) lymphoid structures. A sensitivity analysis of the model’s parameters was also performed. Main results . For H1, H2 and H3 cases respectively, the irradiated fraction of lymphocytes was 99.8 ± 0.7%, 40.4 ± 10.2% et 97.6 ± 2.5%, and the average dose to irradiated pool was 309.9 ± 74.7 mGy, 52.6 ± 21.1 mGy and 265.6 ± 48.5 mGy. The recirculation process considered in the H2 case implied that irradiated lymphocytes were irradiated in the field only 1.58 ± 0.91 times on average after treatment. The OOF irradiation of H&N lymphoid structures considered in H3 was an important contribution to lymphocytes dose. In all cases, the estimated doses are low compared with lymphocytes radiosensitivity, and other mechanisms could explain high prevalence of RIL in patients with brain tumors. Significance . Our framework is the first to take into account OOF doses and recirculation in lymphocyte dose assessment during brain irradiation. Our results demonstrate the need to clarify the indirect effects of irradiation on lymphopenia, in order to potentiate the combination of radio-immunotherapy or the abscopal effect

Synergy of Radiotherapy and a Cancer Vaccine for the Treatment of HPV-Associated Head and Neck Cancer

There is growing interest in the association of radiotherapy and immunotherapy for the treatment of solid tumors. Here, we report an extremely effective combination of local irradiation (IR) and Shiga Toxin B (STxB)-based human papillomavirus (HPV) vaccination for the treatment of HPV-associated head and neck squamous cell carcinoma (HNSCC). The efficacy of the irradiation and vaccine association was tested using a model of HNSCC obtained by grafting TC-1/luciferase cells at a submucosal site of the inner lip of immunocompetent mice. Irradiation and the STxB-E7 vaccine acted synergistically with both single and fractionated irradiation schemes, resulting in complete tumor clearance in the majority of the treated mice. A dose threshold of 7.5 Gy was required to elicit the dramatic antitumor response. The combined treatment induced high levels of tumor-infiltrating, antigen-specific CD8(+) T cells, which were required to trigger the antitumor activity. Treatment with STxB-E7 and irradiation induced CD8(+) T-cell memory, which was sufficient to exert complete antitumor responses in both local recurrences and distant metastases. We also report for the first time that a combination therapy based on local irradiation and vaccination induces an increased pericyte coverage (as shown by αSMA and NG2 staining) and ICAM-1 expression on vessels. This was associated with enhanced intratumor vascular permeability that correlated with the antitumor response, suggesting that the combination therapy could also act through an increased accessibility for immune cells. The combination strategy proposed here offers a promising approach that could potentially be transferred into early-phase clinical trials

An international phase II trial and immune profiling of SBRT and atezolizumab in advanced pretreated colorectal cancer

Abstract Background Immuno-radiotherapy may improve outcomes for patients with advanced solid tumors, although optimized combination modalities remain unclear. Here, we report the colorectal (CRC) cohort analysis from the SABR-PDL1 trial that evaluated the PD-L1 inhibitor atezolizumab in combination with stereotactic body radiation therapy (SBRT) in advanced cancer patients. Methods Eligible patients received atezolizumab 1200 mg every 3 weeks until progression or unmanageable toxicity, together with ablative SBRT delivered concurrently with the 2nd cycle (recommended dose of 45 Gy in 3 fractions, adapted upon normal tissue tolerance constraint). SBRT was delivered to at least one tumor site, with at least one additional measurable lesion being kept from the radiation field. The primary efficacy endpoint was one-year progression-free survival (PFS) rate from the start of atezolizumab. Sequential tumor biopsies were collected for deep multi-feature immune profiling. Results Sixty pretreated (median of 2 prior lines) advanced CRC patients (38 men [63%]; median age, 59 years [range, 20–81 years]; 77% with liver metastases) were enrolled in five centers (France: n = 4, Spain: n = 1) from 11/2016 to 04/2019. All but one (98%) received atezolizumab and 54/60 (90%) received SBRT. The most frequently irradiated site was lung (n = 30/54; 56.3%). Treatment-related G3 (no G4-5) toxicity was observed in 3 (5%) patients. Median OS and PFS were respectively 8.4 [95%CI:5.9–11.6] and 1.4 months [95%CI:1.2–2.6], including five (9%) patients with PFS > 1 year (median time to progression: 19.2 months, including 2/5 MMR-proficient). Best overall responses consisted of stable disease (n = 38; 64%), partial (n = 3; 5%) and complete response (n = 1; 2%). Immune-centric multiplex IHC and RNAseq showed that SBRT redirected immune cells towards tumor lesions, even in the case of radio-induced lymphopenia. Baseline tumor PD-L1 and IRF1 nuclear expression (both in CD3 + T cells and in CD68 + cells) were higher in responding patients. Upregulation of genes that encode for proteins known to increase T and B cell trafficking to tumors (CCL19, CXCL9), migration (MACF1) and tumor cell killing (GZMB) correlated with responses. Conclusions This study provides new data on the feasibility, efficacy, and immune context of tumors that may help identifying advanced CRC patients most likely to respond to immuno-radiotherapy. Trial registration EudraCT N°: 2015–005464-42; Clinicaltrial.gov number: NCT02992912