The FARF Symposium was held in a virtual four-part series format during the month of July 2021. Fanconi anemia (FA) researchers and clinicians met virtually to share updates on specific topics including gene therapy, bone marrow failure, DNA repair, and cancer. The chairs for each session provided an overview of the research presented and insight on the future of the field. Thank you to all sessions chairs for leading excellent sessions and providing the community with these thorough summaries.
Gene Therapy & Gene Editing
Co-chairs: Paula Río and Juan Bueren (both at CIEMAT/CIBERER/Fundación Jiménez Díaz, Spain)
The first day of the Symposium reviewed progress in the FA gene therapy and gene editing fields, with a particular focus on the application of these innovative therapies for the correction of bone marrow failure in FA patients.
Dr. Juan Bueren, a principal investigator of the FANCOLEN I and FANCOLEN II gene therapy trials, shared results from eight evaluable patients that have been treated in the FANCOLEN I trial in Spain. In this trial, six patients have shown progressive engraftment of corrected cells in the bone marrow and peripheral blood, and in two cases, stabilization or even increased peripheral blood cells have been observed at up to five years post treatment with the corrected cells.
Dr. Julian Sevilla (Hospital Niño Jésus) showed the impact of the drug eltrombopag in two patients treated with gene therapy in the FANCOLEN I trial. His data demonstrated that eltrombopag increases the potential for cells to grow in the bone marrow and peripheral blood, suggesting the potential relevance of this drug for improving gene therapy in FA patients.
Dr. Agneiszka Czechowicz (Stanford) and Dr. Jonathan Schwartz (Rocket Pharma) reviewed the progress of the global FANCOLEN II trial. In this trial, FA patients were treated with stem cells corrected by gene therapy in earlier stages of bone marrow failure, as compared to patients in the FANCOLEN I trial. Performing gene therapy at an earlier stage of bone marrow failure enables better collection of stem cells from the patients, which results in higher numbers of infused corrected stem cells. In this trial, six of the seven treated patients who had more than six months of follow up are showing evidence of engraftment. Dr. Schwartz also indicated that five additional patients will be enrolled in the coming months. Importantly, severe side effects have not been observed in any patients enrolled in the FANCOLEN I or FANCOLEN II trials.
The second session was dedicated to recent advances in the field of gene editing for FA. This new approach is a recent alternative to lentiviral-mediated gene therapy that allows the targeted correction of disease-associated mutations. As an example of the clinical application of gene editing, Dr. Sandeep Soni (UCSF) presented the results of the first gene editing clinical trial in patients with beta-thalasemia and sickle cell anemia. Because of this genetic manipulation in the hematopoietic stem cells of the patients, most of the treated patients are no longer dependent on transfusions. Although not yet tested in the clinic, new gene editing technologies were reported to efficiently correct specific FA gene mutations in human hematopoietic stem cells in talks presented by Drs. Jacob Corn (ETH Zurich), Branden Moriarty (University of Minnesota), and Paula Rio (CIEMAT Madrid).
To conclude the gene editing session, Dr. David Liu (Harvard) presented impressive experimental studies showing the efficacy of in vivo Base Editing (BE) in a mouse model of progeria (a rare genetic disease that causes children to age rapidly). Base editing is a CRISPR-Cas9-based genome editing technology. In this model, mice treated with the BE technology showed a correction of the clinical hallmarks of the aging disease. Dr. Liu also showed the efficacy of BE for the ex vivo correction of the beta-globin (protein in red blood cells) in human hematopoietic stem cells from animal models and patients with inherited blood disorders. The studies presented by Dr. Liu demonstrate the potential of gene editing in correcting anemia and clinical manifestations of other diseases, which are important advancements that may be applicable to FA.
Overall, the session on gene therapy revealed unprecedented results in gene therapy in different diseases, including FA. So far, most of the FA patients treated with gene therapy have shown progressive engraftment of corrected stem cells, with an absence of any conditioning regimen or severe side effects. The long-term follow-up of these patients will confirm the use of gene therapy to prevent, stabilize, or reverse bone marrow failure, and perhaps minimize the risk of hematologic cancers. Additionally, new experimental and clinical data show that gene editing may be a potential therapeutic strategy that can be used in the future to correct specific mutations in FA patients.
Bone Marrow Failure
Co-chairs: Sharon Cantor (UMass Medical School) and Peter Kurre (Children’s Hospital of Philadelphia Research Institute)
Speakers and participants from Europe, the United States and Asia shared research outcomes on biology, model systems, and therapy for FA-associated bone marrow failure (BMF) in three separate sessions.
The opening session provided strong evidence that the mechanisms underlying bone marrow failure (BMF) in FA are unlikely to result from loss of DNA repair alone. The first of two keynote speakers, Dr. Filippo Rosselli (Gustave Roussy Institute), discussed evidence that selective loss of FA proteins generates defects in ribosomes (which make proteins necessary for cell growth), already known to cause BMF in related disorders. Work by Dr. Lei Li (Zhejiang University), the second keynote speaker, and Dr. Grant Rowe (Boston Children’s Hospital), indicated that the process by which stem cells generate mature cell populations (differentiation) causes cellular stress and loss of stem cells in part by generating aldehyde metabolites.
The talks from this first session suggest that other core cellular functions beyond DNA repair are impacted by loss of FA protein function, and more insight can be expected from the analysis of FA protein function.
Dr. Andre Larochelle, a featured keynote speaker from the National Institutes of Health, discussed early results from an ongoing clinical trial of the drug eltrombopag in FA patients. Previous non-FA studies had suggested a role for the drug in hematopoietic stem cell self-renewal, which was an important finding that helped establish the rationale to develop this trial specifically for FA patients. Exciting early results from the FA trial suggest potential benefit in halting the progression of FA and even restoration of stem cells.
Dr. Larochelle was followed by a series of talks selected from abstracts with more clinical perspectives. Dr. Jean Soulier (Saint-Louis Hospital) showed that certain abnormalities common in FA (gain in chromosome 1q) indirectly reduce the DNA damage response that is considered the critical barrier for preventing clonal evolution to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). The results from his laboratory group suggest that gains in chromosome 1q in FA patients may be an important clinical biomarker for progression to MDS.
Dr. Yvonne Behrens (Hannover Medical School) also discussed results on MDS risk-based grouping of FA patients. Her team developed a new system to classify the risk of progression in FA patients based on hematologic, bone marrow, and cytogenetic status. This step wise progression model will hopefully be useful in distinguishing abnormalities that require urgent action from those that can be observed.
A theme throughout the day was that the core problems in FA that lead to BMF remain to be discovered, but ongoing studies continue to expand the potential targets to counterbalance FA deficiency and mitigate BMF.
DNA Repair
Chair: Agata Smogorzewska (The Rockefeller University)
Failure to properly repair DNA strands that become linked by strong chemical bonds (interstrand crosslinks) is a hallmark of Fanconi anemia. Therefore, it is fitting that an entire day of the 2021 Symposium was dedicated to learning more about how FA proteins repair DNA in a meeting titled “When Watson and Crick get linked: origins and repair of DNA interstrand crosslinks.” To understand FA as a disease, it is essential to know how these DNA linkages form and how they are repaired. In this year’s Symposium, we heard from numerous speakers about discoveries in the FA DNA repair field that have been made in their laboratories over the past year.
What are the sources that cause DNA interstrand crosslinks? We have known for decades that chemotherapeutics cause DNA interstrand crosslinks, but recent work from Dr. KJ Patel’s laboratory (University of Oxford) has shown that our cells produce aldehydes, chemicals that react with DNA to form interstrand crosslinks. Dr. Patel showed evidence that formaldehyde produced by bone marrow stem cells can overwhelm the cells and cause DNA damage even with a fully functional FA pathway. Working together with Dr. Minoru Takata at the Kyoto University in Japan, they described how individuals who could not detoxify formaldehyde developed disease very similar to FA despite intact FA proteins. This work is transformative and shows a new paradigm that explains the mechanism of disease in FA patients, and highlights a potential new avenue for therapeutic or prevention strategies for bone marrow failure in FA patients.
Bacteria that colonize our bodies, commonly referred to as the microbiome, are also known to produce chemicals that could cause DNA interstrand crosslinks. Dr. Emily Balskus (Harvard University) and Dr. Silvia Balbo (University of Minnesota) showed that a chemical called colibactin made by certain E. coli strains can create DNA interstrand crosslinks. Colibactin has been linked to colorectal cancer, which raises the question of whether this or other bacterial toxins could play a role in head and neck cancer in FA patients. Our understanding of the microbiome in FA patients is still in its infancy, but it is an important area to develop as we work to understand sources of DNA damage in their cells.
How are DNA interstrand crosslinks repaired by the FA pathway? In 2022, we will mark the 30th anniversary of the discovery of the first FA gene, FANCC, by Dr. Manuel Buchwald and colleagues. In the intervening period, a collaboration between FA families and scientists resulted in an almost complete list of genes which, when mutated, lead to FA. We have also learned that the proteins coded by these genes work together to repair DNA interstrand crosslinks and studies from many laboratories elucidated the order of events during the repair. The key step during this process is to place a small protein, called ubiquitin, on FANCI and FANCD2 proteins (the ID complex). This ubiquitination reaction is performed by a set of proteins called the FA core complex. Without the ubiquitination of the ID complex, the DNA repair cannot progress properly, causing disease.
In the last two years, the field took a leap in understanding how ubiquitination regulates the function of the ID complex. This was achieved due to a revolution in cryo-electron microscopy (cryo-EM), which allows for taking snapshots of large protein complexes while they are performing their cellular functions. Dr. Lori Passmore (MRC Laboratory of Molecular Biology) described the overall structure of the core complex at last year’s Symposium. This year, Dr. Nikola Pavletich, (Memorial Sloan Kettering Center) showed the detailed view of the FA core complex in the midst of ubiquitinating the ID complex. He described how the FA proteins in the core complex are necessary for controlling the ubiquitination reaction that takes place on DNA and results in the ID complex encircling the DNA. The structures he presented are extremely informative and will catalyze more research into the basic mechanism of DNA interstrand crosslink repair.
One unanswered question in the field is how the core complex that ubiquitinates the FANCI and FANCD2 proteins is activated. Although we do not understand this step at the structural level yet, Andrew Deans from St. Vincent’s Institute in Australia presented compelling data that showed branched – but not linear DNA – binds a complex of proteins that include FANCM, which in turn, brings in the core complex to the DNA and stimulates its function.
The process of de-ubiquitinating or removing the ubiquitin from the ID complex is equally as important as ubiquitination. It signals that the repair is completed and frees the ID complex from the DNA to allow the next cycles of repair. Dr. Helen Walden (University of Glasgow) described studies which used cryo-EM to reconstruct how USP1 and UAF1 proteins perform the de-ubiquitination reaction. She showed how the interaction between UAF1 and FANCI is necessary for this process, highlighting that we need to improve our understanding of the regulation of this step in DNA repair.
Speakers described progress in the understanding of FA proteins that are called to action once the ID complex is ubiquitinated, including functions of REV7/FANCV and SLX4/FANCP. New functions of FA proteins outside of the canonical pathway of DNA interstrand crosslink repair were also discussed. Dr. Samuel Sidi (Icahn School of Medicine) identified the FANCI protein as an influencer of cell death. Dr. Nibal Badra (University of Patras) identified the importance of the FA proteins in cells that are inappropriately making too many copies of their DNA. Both Dr. Sidi and Badra are newcomers to the FA field, which highlights the growing interest in FA science.
Cancer
Co-chairs: Susanne Wells and Parinda Mehta (University of Cincinnati)
The final day of the Symposium was moderated by Drs. Parinda Mehta and Suzanne Wells and included three sessions focused on FA solid tumors. One of the biggest challenges in the field of FA is our gap in knowledge on the natural history and biology of FA squamous cell carcinomas (SCC). Understanding the fundamental biological mechanisms of FA SCC is necessary to develop approaches for the prevention and treatment of these tumors. It is more critical than ever that we expand this knowledge and focus on the rapid development of nontoxic methods to prevent and treat FA solid tumors.
Research presented on FA solid tumors focused on three broad topics:
- surveillance and screening for premalignancies and surgical approaches;
- new FA SCC mouse models, cancer genomics, and FA protein regulation; and
- novel immune-based and non-cytotoxic therapies.
The first session of the day featured an overview by Dr. Kutler (Weill Cornell Medical Center) on the clinical challenges in the surgical management of HNSCC in patients with FA. Patients with FA often present at a late stage of disease that requires complex surgeries and have a high risk of recurrence. The two talks that followed Dr. Kutler’s were focused on using oral brush biopsy screening as an important way to detect oral cancer early in patients with FA, which would reduce the need for extensive surgery since cancer would be diagnosed at an earlier stage. Madhurima Datta (BC Cancer and University of British Columbia) reported results on a brush biopsy method that focuses on chromosome instability that could be useful for detecting cancer in high-risk patients, such as those with FA. In past years at the FARF Symposium, Dr. Velleuer (Heinrich Heine University) has presented her work on a 14-year study that demonstrated the high efficacy and specificity of a brush biopsy procedure followed by conventional cytology and/or DNA cytometry to test visible lesions in patients with FA. At this year’s Symposium, she presented work on developing self-examination and education materials, including an interactive mobile app that will empower FA patients and their physicians to engage in early screenings.
In the second session, Dr. Ophir Klein (UCSF) presented a review of the normal oral epithelial tissue wherein FA cancers arise and Drs. Markus Grompe (OHSU) and Ramon Garcia-Escudero (CIEMAT) reported on mouse models of FA SCC. An important new finding by Dr. Garcia-Escudero was the observation that mice that don’t express the FANCA gene are prone to pre-malignant and malignant oral lesions. Dr. Agata Smogorzewska (The Rockefeller University) presented genome sequencing data from human FA tumors which indicated a high number of structural variants.
Finally, the third session of the day featured Drs. Robert Ferris (University of Pennsylvania Medical Center) and Stephen Gottschalk (St. Jude’s) who reviewed immune and cellular therapies by checkpoint inhibition and CAR-T cells, respectively. Anecdotal reports of checkpoint inhibitor use in patients with FA with SCC are available, but more objective data from well-thought-out prospective clinical trials will aid in clearly defining their role. Such therapies provide hope and may turn out to be effective cancer treatments in FA.
Dr. Panigraphy (Harvard) reported on studies focused on the role of inflammation in FA cancer as a stimulator of immunotherapy. Drs. Gary Kupfer (Georgetown), Khashayar Roohollahi (Amsterdam UMC), Muhammad Rahman (Barts) and Jordi Surrallés (St. Pau Hospital) presented data supporting potential cancer therapies via inhibitors of mitotic regulators, immune response pathways, and epidermal growth factor receptor (EGFR) signaling. Dr. Jordi Surrallés is currently planning to develop a clinical trial to test the EGFR inhibitor, afatinib, as a treatment for FA SCC.
Tremendous progress has been made regarding early diagnosis, development of novel mouse models, and identification of molecular targets and therapeutic approaches for FA SCC. A common theme that emerged from the research presented in this session and in panel discussions centered around the critical need to identify new targeted strategies that are biology driven and can be safely used for prevention and/or treatment of these cancers in individuals with FA.
