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3149 Loss of Complement Factor I (Cfi) a Negative Regulator of Complement Cascade Activity Exacerbates JAK2V617F-Dependent Phenotype

Program: Oral and Poster Abstracts
Session: 631. Myeloproliferative Syndromes and Chronic Myeloid Leukemia: Basic and Translational: Poster II
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Diseases, immunology, Myeloid Malignancies, Biological Processes, molecular biology, pathogenesis
Sunday, December 10, 2023, 6:00 PM-8:00 PM

Alissa Oakes, BS1*, Dennis M Bonal, MSc, BSc2, Zoe Creane1*, Hyunju Oh, BS3*, Anna Dorota Chorzalska, PhD4*, Brooke Sadler, PhD5, John L. Reagan, MD6,7, Rabin Niroula8*, Adam J Olszewski, MD1,9,10,11, Gabriel Haller, PhD12*, Stephen Donnelly, BS13* and Patrycja M Dubielecka, PhD14,15*

1Brown University, Providence, RI
2Pathobiology Graduate Program, Brown University, Douglas, MA
3Warren Alpert Medical School, Brown University, Providence, RI
4Signal Transduction Lab, Rhode Island Hospital, Providence, RI
5Pediatrics, Washington University School of Medicine, Saint Louis, MO
6Legorreta Cancer Center of Brown University, Providence, RI
7Warren Alpert Medical School of Brown University, Providence, RI
8Lifespan Cancer Institute, The Warren Alpert Medical School of Brown University, Providence, RI
9Lifespan Cancer Institute, Warren Alpert Medical School of Brown University, Providence, RI
10Lifespan Cancer Institute, Providence, RI
11Legorreta Cancer Center at Brown University, Lifespan Cancer Institute, Providence, RI
12Washington University at St. Louis, St. Louis, MO
13Lifespan Oncology Clinical Research, Rhode Island Hospital, Providence, RI
14Rhode Island Hospital and Warren Alpert Medical School At Brown University, Providence, RI
15Pathobiology Graduate Program, Brown University, Providence, RI

Background: The current evidence suggests that inflammatory phenotype contributes to the pathogenesis of myeloproliferative neoplasms (MPNs). While our understanding of complement cascade contributions to inflammation-mediated pathologies has expanded, role this cascade plays in etiology of myeloid malignancies has not been detailed. Previously, we uncovered an inactivating RNSV in complement factor I (CFI G119R), an inhibitor of the complement cascade, in 20% of PMF patients (n=10) using WGS, and in a separate patient cohort (n=10) we observed an increase in CH50 levels (n=3) and decrease in C3 levels (n=3) (Oakes et al., 2021, Blood, 138:1472).These findings prompted us to generate murine models allowing assessment of the Cfi loss-driven complement overactivity effects on hematopoiesis and development of MPNs.

Methods: A conventional Cfi knockout (KO) mouse model was generated by targeting exon 2 of the Cfi gene using CRISPR/Cas9. Inactivation of one or two copies of Cfi was confirmed by genotyping and total loss (Cfi KO) or Cfi deficiency (Cfi HET) were confirmed in peripheral blood (PB) plasma. Complete blood counts (CBC) and the frequencies of granulocytes, B cells, plasmocytes, T cells, macrophages/monocytes, platelets, erythroid cells in the bone marrow were longitudinally assessed by flow cytometry for 40 weeks in Cfi WT, HET and KO mice. Cfi HET animals were crossed with JAK2V617F(fl/+);Mx1-cre(+) mice (obtained from crossing B6.Cg-Tg(Mx1-cre+ 1)Cgn/J; # 003556, JAX) and JAK2V617F expressing (B6N.129S6(SJL)-Jak2tm1.2Ble/AmlyJ #031658, JAX) to generate Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre(+/-) (CFI/JAK2) strain. Complete blood counts and survival were monitored for Cfi(+/+)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFIWT/JAK2HET) Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFIHET/JAK2HET) or Cfi(-/-)/JAK2V617F(fl/+); Tg(Mx1-cre)(+/-) (CFIKO/JAK2HET) mice.

Results: Cfi HET and KO mice were fertile and viable. Cohort of Cfi WT n=13, Het n=12, KO n=13 mice was subjected to analyses. Genotypes were confirmed via PCR and western blot. Flow cytometry demonstrated a copy loss dependent decrease in total T-cells (CD3+) and increase in mature resting B-cells (CD19+/B220+). CBC demonstrated a copy loss dependent decrease in both monocyte (p adj. KO vs. WT 0.002) and granulocyte percentage (p adj. KO vs. WT 0.003), and increase in lymphocyte percentage (p adj. KO vs. WT 0.001). In PB plasma we detected increased C3 activation in KO mice compared to WT and Het, demonstrated as decreased C3 alpha and presence of inactivated C3 due to increased proteolytical processing of C3 complex. Proteolytic cleavage of CFB compared to Cfi WT and Het was also noted in Cfi KO animals. A significant decrease in survival probability of CFIHET/JAK2HET vs. CFIWT/JAK2HET (p=0.03), CFIKO/JAK2HET vs. CFIWT/JAK2HET (p=0.003), and CFIHET/JAK2HET vs. CFIKO/JAK2HET (p=0.05) averaging 92 days for CfiHET/JAK2HET, 67 days for CfiKO/JAK2HET vs. 98 days for CfiWT/JAK2HET was noted (Fig. A). A granulocyte and monocyte percentage of CFIHET/JAK2HET was increased as compared to CFIWT/JAK2HET mice (Fig. B).

Conclusions: An overactivation of complement cascade at the C3 level is noted in Cfi deficient animals, that is phenotypically linked to an increase in frequency of B cells. These findings may suggest involvement of Cfi directly, and/or in conjunction with generally overactive complement, in B-cell maturation and function. The absence of one copy of Cfi in JAK2V617F mice shows more severe MPN disease progression resulting in markedly decreased survival, linked to increased frequencies of monocytes and granulocytes and markedly decreased lymphocytes. In sum, these data indicate that increased complement activity may increase JAK2V617F-dependent phenotype severity. In depth mechanistic studies detailing observed phenotype are warranted.

Disclosures: Reagan: Pfizer: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees. Olszewski: Leukemia & Lymphoma Society, Genetech, Inc. / F. Hoffmann-La Roche Ltd, Adaptive Biotechnologies, Precision Biosciences, Genmab: Research Funding; Genmab, Blue Cross/Blue Shield of Rhode Island, Schrodinger, ADC Therapeutics, BeiGene: Consultancy.

*signifies non-member of ASH