The emergence of drug resistance limits the efficacy of targeted therapies in human tumors. The prevalent view is that resistance is a fait accompli: when treatment is initiated, cancers already ...contain drug-resistant mutant cells. Bacteria exposed to antibiotics transiently increase their mutation rates (adaptive mutability), thus improving the likelihood of survival. We investigated whether human colorectal cancer (CRC) cells likewise exploit adaptive mutability to evade therapeutic pressure. We found that epidermal growth factor receptor (EGFR)/BRAF inhibition down-regulates mismatch repair (MMR) and homologous recombination DNA-repair genes and concomitantly up-regulates error-prone polymerases in drug-tolerant (persister) cells. MMR proteins were also down-regulated in patient-derived xenografts and tumor specimens during therapy. EGFR/BRAF inhibition induced DNA damage, increased mutability, and triggered microsatellite instability. Thus, like unicellular organisms, tumor cells evade therapeutic pressures by enhancing mutability.
Immune checkpoint inhibitors (CPIs) represent an effective therapeutic strategy for several different types of solid tumors and are remarkably effective in mismatch repair deficient (MMRd) tumors, ...including colorectal cancer (CRC). The prevalent view is that the elevated and dynamic neoantigen burden associated with the mutator phenotype of MMRd fosters enhanced immune surveillance of these cancers. In addition, recent findings suggest that MMRd tumors have increased cytosolic DNA, which triggers the cGAS STING pathway, leading to interferon-mediated immune response. Unfortunately, approximately 30% of MMRd CRC exhibit primary resistance to CPIs, while a substantial fraction of tumors acquires resistance after an initial benefit. Profiling of clinical samples and preclinical studies suggests that alterations in the Wnt and the JAK-STAT signaling pathways are associated with refractoriness to CPIs. Intriguingly, mutations in the antigen presentation machinery, such as loss of MHC or Beta-2 microglobulin (B2M), are implicated in initial immune evasion but do not impair response to CPIs. In this review, we outline how understanding the mechanistic basis of immune evasion and CPI resistance in MMRd CRC provides the rationale for innovative strategies to increase the subset of patients benefiting from CPIs.
Neoantigens that arise as a consequence of tumor-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumor types, a ...high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time.
We performed whole exome sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and in vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures, and predicted neoantigens.
The majority of CRC models showed remarkably stable mutational and neoantigen profiles; however, those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly evolving CRC.
These results indicate that CRCs carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.
Mismatch repair (MMR) protein expression in colorectal cancer (CRC) cells is usually homogeneously retained or lost. Rare lesions may show a heterogeneous pattern of MMR protein expression. We ...evaluated MMR protein expression (MLH1, MSH2, MSH6, and PMS2) in 200 CRCs, identifying 3 groups with proficient MMR protein expression (MMRp), deficient MMR protein expression (MMRd), and heterogeneous MMR protein expression (MMRh). MMRh tumors were microdissected on the basis of the expression of the heterogeneous marker. DNA was extracted and subjected to targeted sequencing. RNA was purified from bulk tumors of all MMRh cases and in a control series of 15 MMRp and 10 MMRd CRCs and analyzed using the PanCancer IO 360 Panel (NanoString Technologies). Twenty-nine of the 200 cases (14.5%) were MMRd. Nine cases (4.5%) showed a heterogeneous pattern of MMR expression, with 6 tumors harboring concomitant loss of one of the other MMR proteins, thus featuring areas with double loss at immunohistochemistry (IHC) testing (MMRh double-loss cases). Four of the 6 MMRh double-loss cases were suitable for a separate sequence variant analysis of IHC double-negative and IHC single-negative components of the tumor. In all lesions, both components exhibited a high tumor mutation burden (TMB). Nevertheless, a significant increase in TMB in the double-negative components was observed (mean TMB: negative, 70 mut/Mb vs positive, 59 mut/Mb) because of a higher number of subclonal variants compared with the other component. Comparative gene expression analyses among MMRd, MMRp, and MMRh CRCs highlighted differential gene expression patterns and an increased number of tumor-infiltrating lymphocytes in MMRh lesions, which is also characterized by a substantial population of exhausted CD8+ lymphocytes. We describe a unique subgroup of CRCs showing heterogeneous expression of MMR proteins in a background of concomitant loss of one of the other markers.
Inactivation of beta-2 microglobulin (B2M) is considered a determinant of resistance to immune checkpoint inhibitors (ICPi) in melanoma and lung cancers. In contrast,
loss does not appear to affect ...response to ICPis in mismatch repair-deficient (MMRd) colorectal tumors where biallelic inactivation of
is frequently observed. We inactivated
in multiple murine MMRd cancer models. Although MMRd cells would not readily grow in immunocompetent mice, MMRd
null cells were tumorigenic and regressed when treated with anti-PD-1 and anti-CTLA4. The efficacy of ICPis against MMRd
null tumors did not require CD8
T cells but relied on the presence of CD4
T cells. Human tumors expressing low levels of B2M display increased intratumoral CD4
T cells. We conclude that B2M inactivation does not blunt the efficacy of ICPi in MMRd tumors, and we identify a unique role for CD4
T cells in tumor rejection. SIGNIFICANCE:
alterations, which impair antigen presentation, occur frequently in microsatellite-unstable colorectal cancers. Although in melanoma and lung cancers
loss is a mechanism of resistance to immune checkpoint blockade, we show that MMRd tumors respond to ICPis through CD4
T-cell activation.
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Patients affected by colorectal cancer (CRC) with DNA mismatch repair deficiency (MMRd), often respond to immune checkpoint blockade therapies, while those with mismatch repair-proficient (MMRp) ...tumors generally do not. Interestingly, a subset of MMRp CRCs contains variable fractions of MMRd cells, but it is unknown how their presence impacts immune surveillance. We asked whether modulation of the MMRd fraction in MMR heterogeneous tumors acts as an endogenous cancer vaccine by promoting immune surveillance. To test this hypothesis, we use isogenic MMRp (Mlh1+/+) and MMRd (Mlh1−/−) mouse CRC cells. MMRp/MMRd cells mixed at different ratios are injected in immunocompetent mice and tumor rejection is observed when at least 50% of cells are MMRd. To enrich the MMRd fraction, MMRp/MMRd tumors are treated with 6-thioguanine, which leads to tumor rejection. These results suggest that genetic and pharmacological modulation of the DNA mismatch repair machinery potentiate the immunogenicity of MMR heterogeneous tumors.
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•MMR heterogeneity affects immune surveillance in mouse CRC•In vivo 6TG treatment increases the MMRd fraction of MMR heterogeneous tumors•Pharmacological selection of MMRd cells improves immune surveillance in MMR tumors
The impact of MMR heterogeneity on immune surveillance of colon cancers is largely unexplored. This proof-of-concept study by Amodio et al. suggests that MMR heterogeneity can be exploited through an endogenous vaccination approach based on pharmacological modulation of the DNA MMR, which potentiates cancer immunogenicity.
Most patients with
-mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRAS
inhibition, whereas patients with colorectal cancer bearing the same mutation rarely ...respond. To investigate the cause of the limited efficacy of KRAS
inhibitors in colorectal cancer, we examined the effects of AMG510 in
colorectal cancer cell lines. Unlike NSCLC cell lines,
colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRAS
inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRAS
blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRAS
inhibitors. The combinatorial targeting of EGFR and KRAS
is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with
colorectal cancer. SIGNIFICANCE: The efficacy of KRAS
inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRAS
inhibition in colorectal cancer. EGFR and KRAS
should be concomitantly inhibited to overcome resistance to KRAS
blockade in colorectal tumors.
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Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise ...heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that
, and
mRNA expression characterized rare tumor-infiltrating T cells.
expression of the transcripts was increased in coisogenic
-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT
RAG1/2
cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.
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