Despite the loss of Adenomatous Polyposis Coli (APC) in a majority of colorectal cancers (CRC), not all CRCs bear hallmarks of Wnt activation, such as nuclear β-catenin. This underscores the presence of other Wnt regulators that are important to define, given the pathogenic and prognostic roles of nuclear β-catenin in human CRC. Herein, we investigated the effect of Casitas B-lineage lymphoma (c-Cbl) on nuclear β-catenin, which is an oncoprotein upregulated in CRC due to loss-of-function APC or gain-of-function CTNNB1 mutations. Despite mechanistic rationale and recent discoveries of c-Cbl's mutations in solid tumors, little is known about its functional importance in CRC. Our study in a cohort of human CRC patients demonstrated an inverse correlation between nuclear β-catenin and c-Cbl. Further investigation showed that the loss of c-Cbl activity significantly enhanced nuclear β-catenin and CRC tumor growth in cell culture and a mouse xenograft model. c-Cbl interacted with and downregulated β-catenin in a manner that was independent of CTNNB1 or APC mutation status. This study demonstrates a previously unrecognized function of c-Cbl as a negative regulator of CRC.

Beta hemoglobinopathies resulting from dysfunctional or deficient adult beta-globin expression are some of the most prevalent inherited blood disorders in the world. Upregulation of developmentally-silenced fetal gamma-globin would replace adult beta-globin to ameliorate disease symptoms. One of the approaches to reactivate fetal globin expression in erythroid cells is through gene editing by zinc finger or CRISPR-Cas9 nucleases to disrupt the expression of a transcription factor BCL11A, which mediates fetal globin silencing. As BCL11A-deficiency leads to hematopoietic stem cells (HSCs) defects, the current editing approaches target the BCL11A erythroid-enhancer region located in intron 2 of the BCL11A gene to selectively reduce BCL11A expression in erythroid cells. Instead of targeting BCL11A, we sought to identify novel cis-regulatory elements at the beta-globin locus for targeted gene editing to achieve fetal globin reactivation. From a lenti-CRISPR mediated saturated mutagenesis screen covering the beta-globin locus using Human Umbilical Cord Blood-Derived Erythroid Progenitor (HUDEP)-2 cells, multiple fetal hemoglobin (HbF)-inducing genomic domains were identified. Most of the hits were concentrated at the gamma-globin (HBG1/2) promoters, clustered at known hereditary persistence of fetal hemoglobin (HPFH) mutation hotspots. In-depth genotype to phenotype analysis further defined the indels responsible for HbF induction in these subdomains. We interrogated multiple families of nucleases and guide RNA (gRNA) combinations with or without single-stranded oligodeoxynucleotides (ssODN) to guide editing outcome. gRNAs were selected based on their HbF induction potential (up to 40%) when introduced into mobilized peripheral blood (mPB) CD34+ hematopoietic stem and progenitor cells (HSPCs) as ribonucleoprotein (RNP) complexes. HSPCs transfected with RNPs targeting either the BCL11A erythroid-enhancer or the HBG1/2 proximal regions were then injected into NBSGW mice to study the editing in SCID-repopulating cells (SRC) and their multilineage reconstitution potential. All groups achieved high levels of human chimerism (>70% hCD45+/hCD45+mCD45) and comparable monocytes, granulocytes, B lymphocytes, and hCD34+ HSPCs reconstitution. However, BCL11A-edited cells showed selective reduction in erythroid lineage (CD235a+) output, up to 4-fold lower than untreated or HBG1/2 promoter-edited HSPCs. Sequencing analysis from lineage-specific sorted cells further revealed reduced editing levels at BCL11A erythroid-specific enhancer in the erythroid compartment compared to unfractionated bone marrow (BM) or other human lineages (70% in erythroid vs. 90% in all other lineages). Furthermore, the nonproductive fraction of indels that did not disrupt the BCL11A GATAA motif was significantly enriched in erythroid cells (22% in erythroid vs. 8% in other lineages). Ex vivo erythroid cultures suggests BCL11A erythroid-enhancer editing may lead to slightly increased apoptosis during erythroid differentiation. In contrast, HBG1/2 promoter-edited cells had similar editing levels and indel patterns across all lineages with no significant lineage skewing. When chimeric BM from HBG1/2 promoter-edited groups were cultured in erythroid conditions, ex vivo-derived erythroid cells had significantly elevated levels of HbF compared to controls. When CD235a+ cells were sorted without further culture from chimeric BM of mice engrafted with HBG1/2 promoter-edited cells, significantly increased levels of HbF were detected by UPLC (up to 30%) compared to controls (~6%). Thus, long-term HSCs have been edited productively at the HBG1/2 promoters. These cells were able to generate erythroid progenitors that harbor HbF-inducing indels, which in turn, gave rise to erythroid cells in vivo with a clinically-relevant levels of HbF in a xenotransplantation model. Together, our data suggest that BCL11A-edited cells have an erythroid differentiation defect or survival disadvantage in NBSGW mouse model that warrants further investigation. In contrast, editing of the HBG1/2 promoters in mPB CD34+ cells achieved sustained HbF expression in erythroid lineage while maintaining multilineage differentiation potential. Targeting of the HBG1/2 promoters in HSPCs may be an attractive strategy for the development of potential gene editing medicines for beta hemoglobinopathies. Disclosures Chang: Editas Medicine Inc.: Employment, Equity Ownership. Sanchez:Editas Medicine Inc.: Employment, Equity Ownership. Heath:Editas Medicine Inc.: Employment, Equity Ownership. deDreuzy:Editas Medicine Inc.: Employment, Equity Ownership. Haskett:Editas Medicine Inc.: Employment, Equity Ownership. Vogelaar:Editas Medicine Inc.: Employment. Gogi:Editas Medicine Inc.: Employment, Equity Ownership. Da Silva:Editas Medicine Inc.: Employment, Equity Ownership. Wang:Editas Medicine Inc.: Employment, Equity Ownership. Sadowski:Editas Medicine Inc.: Employment, Equity Ownership. Gotta:Editas Medicine Inc.: Employment, Equity Ownership. Siwak:Editas Medicine Inc.: Employment, Equity Ownership. Viswanathan:Editas Medicine Inc.: Employment, Equity Ownership. Loveluck:Editas Medicine Inc.: Employment, Equity Ownership. Chao:Editas Medicine Inc.: Employment, Equity Ownership. Tillotson:Editas Medicine Inc.: Employment, Equity Ownership. Chalishazar:Editas Medicine Inc.: Employment, Equity Ownership. Dass:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Brennan:Editas Medicine Inc.: Employment, Equity Ownership. Tabbaa:Editas Medicine Inc.: Employment, Equity Ownership. Marco:Editas Medicine Inc.: Employment, Equity Ownership. Zuris:Editas Medicine Inc.: Employment, Equity Ownership. Reyon:Editas Medicine Inc.: Employment, Equity Ownership. Isik:Editas Medicine Inc.: Employment, Equity Ownership. Friedland:Editas Medicine Inc.: Employment, Equity Ownership. Ta:Editas Medicine Inc.: Employment, Equity Ownership. Harbinski:Editas Medicine Inc.: Employment, Equity Ownership. Giannoukos:Editas Medicine Inc.: Employment, Equity Ownership. Teixeira:Editas Medicine Inc.: Employment, Equity Ownership. Wilson:Editas Medicine Inc.: Employment, Equity Ownership. Albright:Editas Medicine Inc.: Employment, Equity Ownership. Jiang:Editas Medicine Inc.: Employment, Equity Ownership.

Background: Many different treatment regimens are currently available for Multiple Myeloma (MM) patients. There is ongoing research to identify subgroups of patients who may benefit from specific treatments. Recently, it was found that patients with low-risk MM (SKY92 standard-risk, ISS stage I, and no del(17p)) had a better overall survival after receiving Bortezomib, Melphalan and Prednisone (VMP) combination therapy compared to High-dose Melphalan therapy followed by an autologous stem cell transplantation (HDM+ASCT)(Hofste op Bruinink et al. 2018, The American Society of Hematology, # 3186). Clinicians may consider a risk-stratified treatment approach based on SKY92, ISS, and del(17p) status for transplant-eligible MM patients instead of treating all transplant-eligible patients with HDM+ASCT. Aims: We evaluated the cost-effectiveness of a risk-stratified treatment approach for transplant-eligible MM patients in which low-risk individuals are directly treated with combination therapy instead of HDM+ASCT. All others patients will receive HDM+ASCT as initial treatment. Methods: The cost-effectiveness analysis was restricted to the low-risk patient group because risk stratification only led to a therapy change for this subgroup. A decision model was developed consisting of three health states: progression-free survival, progressive disease and death. A partitioned survival model, based on Kaplan-Meier curves reported by Hofste op Bruinink et al. (2018), was used to estimate the proportion of patients in each health state over time. Quality of life utilities and costs were derived from literature. Costs consisted of additional testing costs, initial treatment costs, maintenance treatment costs and costs of progressive disease. It was assumed that all patients received maintenance treatment until disease progression and that second and further-line treatments were identical for both VMP and HDM+ASCT. The main outcome of the analysis was the incremental costs per life year or quality-adjusted life year (QALY) gained. The analysis was performed from a Dutch health care perspective (in 2018 Euros) with a lifetime horizon. Results: 26% of all transplant-eligible patients were classified as lowrisk. In these patients, VMP resulted in better health outcomes compared to HDM+ASCT. The difference in life years was 9.6 years (VMP: 21.3 years, HDM+ASCT: 11.7 years). The QALY gain was 6.4 QALY (VMP: 14.8, HDM+ASCT: 8.4 QALYs). Initial treatment costs were almost similar for the two treatments, but the costs of maintenance treatment and progressive disease were substantially larger for VMP due to a longer life expectancy. The total difference in costs was €345,000 (VMP: €876,245, HDM+ASCT: €531,245). The incremental cost-effectiveness ratio was €35,677 per life year gained and €54,005 per QALY gained. Summary/Conclusion: The proposed risk-stratified treatment approach resulted in substantial survival and QALY gain. However, this health gain is also associated with higher costs due to continuous maintenance treatment until prog...

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