Periodic Reporting for period 1 - AMBER (APOBEC Mutagenesis: a novel Achilles heel of Breast cancer)
Periodo di rendicontazione: 2022-10-01 al 2025-03-31
APOBEC mutagenesis is a cellular mechanism by which genetic alterations are acquired somatically, driven by APOBEC enzyme family members. This mechanism is prominently observed in 15% of primary and 25% of recurrent breast cancer, the most common cause of cancer-related death in middle-aged women. Current evidence suggests that APOBEC mutagenesis contributes to all disease stages, i.e. cancer initiation, progression and treatment resistance. The core idea of the AMBER project is that unravelling the mechanism of APOBEC mutagenesis induction and maintenance will turn this mechanism into breast cancer’s Achilles heel and yielding therapeutic targets. To prove this, I will answer the following challenging research questions: Why is APOBEC mutagenesis operational in breast cancer? How does APOBEC contribute to disease progression? Can we target tumours having APOBEC mutagenesis specifically?
To this end, I will first collect epidemiological and molecular evidence for factors inducing APOBEC mutagenesis in breast cancer. This may help to prevent APOBEC mutagenesis from occurring, potentially decreasing breast cancer incidence. Second, using global and single cell genomics, I will secure a link between APOBEC mutagenesis and disease progression, giving potential leads to delay progression. Third, I will exploit a likely vulnerability of APOBEC driven breast cancer relying on my finding that these tumours may depend on a proficient homologous DNA repair (HR) pathway. When experimentally confirmed, targeting HR may extinguish APOBEC-driven disease. Finally, and building on my observation that APOBEC mutagenesis associates with a profound immune response, I hypothesize that this is due to the generation of a new class of neo-epitopes being produced. If proven true, targeting these neo-epitopes can provide another effective means to eradicate APOBEC driven tumours by immune therapies. In short, I anticipate that this AMBER project will provide the fundamental insights into APOBEC mutagenesis needed to turn it into an Achilles heel which can be targeted to prevent, delay, and ultimately cure APOBEC-driven breast cancer.
To this end, I will first collect epidemiological and molecular evidence for factors inducing APOBEC mutagenesis in breast cancer. This may help to prevent APOBEC mutagenesis from occurring, potentially decreasing breast cancer incidence. Second, using global and single cell genomics, I will secure a link between APOBEC mutagenesis and disease progression, giving potential leads to delay progression. Third, I will exploit a likely vulnerability of APOBEC driven breast cancer relying on my finding that these tumours may depend on a proficient homologous DNA repair (HR) pathway. When experimentally confirmed, targeting HR may extinguish APOBEC-driven disease. Finally, and building on my observation that APOBEC mutagenesis associates with a profound immune response, I hypothesize that this is due to the generation of a new class of neo-epitopes being produced. If proven true, targeting these neo-epitopes can provide another effective means to eradicate APOBEC driven tumours by immune therapies. In short, I anticipate that this AMBER project will provide the fundamental insights into APOBEC mutagenesis needed to turn it into an Achilles heel which can be targeted to prevent, delay, and ultimately cure APOBEC-driven breast cancer.
To find epidemiological evidence why APOBEC mutagenesis is profoundly present in 10-15% of cases with primary breast cancer, we have selected tumors of >500 ER+/HER2- breast cancer patients from HEBON for next generation sequencing to determine APOBEC mutagenesis status. For these cases, we also have self-reported information regarding relevant risk factors allowing us to link APOBEC mutational status to all documented risk factors.
To reveal the gene regulatory network of APOBEC enzymes APOBEC3A (A3A) and APOBECA3B (A3B), we have now successfully implemented single cell CRISPR-Cas9-based gene perturbation in parallel with single-cell RNA sequencing to identify genes that may regulate A3B in the breast cancer cell line MCF7. Via lentiviral transduction, we introduced the gene editing system as well as guides targeting 22 selected candidates that are strongly co-expressed with A3B and performed single cell transcriptome analysis of ~20,000 targeted cells. Detailed bioinformatics analysis revealed 6 novel gene regulatory networks significantly impacting A3B expression of which one network has already been independently validated.
To determine whether breast cancers displaying APOBEC mutagenesis are more aggressive and/or more heterogeneous, we have selected DNA samples from treatment-naïve ER+/HER2- breast cancer patients who were stratified for disease progression. To verify the clinical significance of A3A and A3B at the protein level, we have additionally stained our tissue microarray containing 646 primary breast cancer specimens. While the analysis for A3A is still ongoing, we have observed that A3B protein expression was correlated with clinical outcome.
To study whether the induction of HR deficiency is synthetically lethal in breast cancer cells having APOBEC mutagenesis, we have generated HAP1 clones with and without A3B expression.
Lastly, to identify whether APOBEC mutated breast cancer generates a novel class of neo-epitopes, we have successfully set-up and optimized an immunopeptidome/mass-spectometry workflow. With this workflow, we achieved optimal and efficient immunoprecipitation of peptides presented via the Major Histocompatibility Complex (and thus representing targets for T cells) for 8 cell line samples including the HAP1 clones. In combination with in silico generated databases, we have identified >100 neo-peptides uniquely related to APOBEC mutagenesis.
To reveal the gene regulatory network of APOBEC enzymes APOBEC3A (A3A) and APOBECA3B (A3B), we have now successfully implemented single cell CRISPR-Cas9-based gene perturbation in parallel with single-cell RNA sequencing to identify genes that may regulate A3B in the breast cancer cell line MCF7. Via lentiviral transduction, we introduced the gene editing system as well as guides targeting 22 selected candidates that are strongly co-expressed with A3B and performed single cell transcriptome analysis of ~20,000 targeted cells. Detailed bioinformatics analysis revealed 6 novel gene regulatory networks significantly impacting A3B expression of which one network has already been independently validated.
To determine whether breast cancers displaying APOBEC mutagenesis are more aggressive and/or more heterogeneous, we have selected DNA samples from treatment-naïve ER+/HER2- breast cancer patients who were stratified for disease progression. To verify the clinical significance of A3A and A3B at the protein level, we have additionally stained our tissue microarray containing 646 primary breast cancer specimens. While the analysis for A3A is still ongoing, we have observed that A3B protein expression was correlated with clinical outcome.
To study whether the induction of HR deficiency is synthetically lethal in breast cancer cells having APOBEC mutagenesis, we have generated HAP1 clones with and without A3B expression.
Lastly, to identify whether APOBEC mutated breast cancer generates a novel class of neo-epitopes, we have successfully set-up and optimized an immunopeptidome/mass-spectometry workflow. With this workflow, we achieved optimal and efficient immunoprecipitation of peptides presented via the Major Histocompatibility Complex (and thus representing targets for T cells) for 8 cell line samples including the HAP1 clones. In combination with in silico generated databases, we have identified >100 neo-peptides uniquely related to APOBEC mutagenesis.
At this moment there are two findings that are beyond the state of the art.
First, we have implemented massive gene perturbation combined with single-cell RNA sequencing to chart gene networks that regulate A3B expression where we started from co-expressed genes controlling divergent transcriptional networks. One of the 6 identified networks impacting A3B expression has been validated and constitutes a novel regulator that is now being explored functionally.
Secondly, we have developed an immunopeptidome workflow to collect 8-11-mer breast cancer-derived peptides, which, together with an in-house developed proteome database that covers predicted APOBEC-derived neo-peptides, has already yielded >100 of such neo-peptides. This collection of APOBEC-driven neo-epitopes is further expanded and may constitute promising novel targets for T cell therapies.
First, we have implemented massive gene perturbation combined with single-cell RNA sequencing to chart gene networks that regulate A3B expression where we started from co-expressed genes controlling divergent transcriptional networks. One of the 6 identified networks impacting A3B expression has been validated and constitutes a novel regulator that is now being explored functionally.
Secondly, we have developed an immunopeptidome workflow to collect 8-11-mer breast cancer-derived peptides, which, together with an in-house developed proteome database that covers predicted APOBEC-derived neo-peptides, has already yielded >100 of such neo-peptides. This collection of APOBEC-driven neo-epitopes is further expanded and may constitute promising novel targets for T cell therapies.