Pilar Guerra (José Buchs, 1926)

To explore the malignant potential of the observed phenotype, we transplanted total BM cells from diseased Vav1-iCre;Nsd1fl/fl mice (carrying the isogenic CD45.2+ marker) either alone or in competition with normal CD45.1+ cells into lethally irradiated syngeneic recipients (CD45.1+) (Figure 17A). All transplanted recipients rapidly developed symptoms of disease within 40 days post transplantation and showed hepato-splenomegaly, increased white blood counts, anemia, thrombocytopenia, mild lymphocytosis, and accumulation of LUC and reticulocytosis (Figure 17B-I). In addition, flow cytometry analysis of peripheral blood and BM revealed the accumulation of early erythroid progenitors found in R0 fraction (Figure 17J-K). Chimerism analysis of peripheral blood confirmed presence of injected CD45.2+ cells in peripheral blood (Figure 17L).

We next assessed whether the disease remains transplantable in presence of increasing number of normal competitor cells, and whether it might be possible to further narrow down the disease-initiating cells based on c-Kitand CD71 expression (Figure 18A). First, we checked the colony forming capacity within the Lin-_/c-Kit+

compartment and found that only CD71low _{and CD71}intermediate _{expressing cells of}

Vav1-iCre;Nsd1fl/fl mice had colony forming potential (Figure 18B). We next transplanted sorted Lin-_/c-Kit+ _{and CD71 low/intermediate/high expressing cells or}

whole BM alone or in 1:10 competition (CD45.2+_{) together with equal amounts of}

competitor whole BM (CD45.1+) into lethally irradiated recipients (CD45.1+) (Figure 18C&D). Kaplan- Meier survival curves confirmed development of disease upon transplanting whole knockout BM. Interestingly, all recipients transplanted with BM of diseased mice in 1:10 competition (WT:KO = 10:1) as well as mice receiving CD71low

cells (WT:KO CD71low _{10:1) developed disease. Moreover, 75% of CD71}intermediate

transplanted mice (WT:KO CD71mid 10:1) and only 50% of mice that receiving CD71high _{cells (WT:KO CD71high 10:1) developed disease symptoms with increasing}

time post- transplantation (Figure 18E). All diseased mice showed splenomegaly, increased white blood cell counts, decreased red blood cell counts, thrombocytopenia, variable amounts of reticulocytosis, some lymphocytosis as well as appearance of LUCs (Figure 18F-K, data not shown).

Taken together, these data show that disease in Vav1-iCre;Nsd1fl/fl

mice is transplantable into secondary recipients in competition and the disease initiating cell may be within the in the Lin-/c-Kit+ _{and most probably CD71}-/dim _compartment.

FIGURE 17. Competitive BMT of whole BM cells of diseased Vav1-iCre;Nsd1fl/fl mice. (A) Schematic depiction of competitive transplantation experimental setup. 106 CD45.2+ whole bone marrow cells of Nsd1fl/fl (n=4/”WT 1:0”) or diseased Vav1-iCre;Nsd1fl/fl mice (n=4/”KO 0:1”) were transplanted without competition or 106 CD45.1+ wildtype cells (n=6/”WT:KO 1:1”) into lethally irradiated CD45.1+ recipients. (B) Kaplan Meier survival curve of whole bone marrow transplanted mice. Black line represents WT 1:0 group that received bone marrow of Nsd1fl/fl. Red line represents KO 0:1 group that received bone marrow of diseased Vav1-iCre;Nsd1fl/fl mice. Orange Line represents competitive transplantation group that received bone marrow from control and diseased Vav1-iCre;Nsd1fl/fl mice in a 1:1 ratio (WT:KO 1:1). Survival is shown as percentage at days after transplantation. Median survival of KO 0:1 transplanted mice was 33 days and 42 days of WT:KO 1:1 mice. WT 1:0 group was analyzed at 42 days. (C) Spleen and liver weight of transplanted mice in gram: WT 1:0 (n=2, black bar), KO 0:1 (n=4, red bar), WT:KO 1:1 (n=6, orange bar) (Students t-test with unequal variances. *** p<0.001, **** p<0.0001). (D) Peripheral blood counts of white blood cells, (E) red blood cells, (F) platelets, (G) lymphocytes, (H) monocytes (MONO), eosinophils (EOS), basophils (BASO), neutrophils (NEO), “large unstained cells” (LUC) and (I) reticulocytes in WT 1:0 (n=3, black bar), KO 0:1 (n=2, red bar), WT:KO 1:1 (n=5, orange bar) groups (Students t-test with unequal variances. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). (J) Percentage flow cytometry marker stained cell population in unlysed peripheral blood and (K) bone marrow at day of sacrifice in WT 1:0 (n=2, black bar), KO 0:1 (n=4, red bar), WT:KO 1:1 (n=6, orange bar) to distinguish maturation steps in erythropoiesis: R0 fraction (CD71-/dim, Ter119-), R1 (CD71+, Ter119-), R2 (CD71+, Ter119+), R3 (CD71+/dim, Ter119+) and R4 (CD71-, Ter119+) (Two-way ANOVA with Turkeys multiple comparisons. *** p<0.001, **** p<0.0001). (L) Percentage flow cytometry marker stained cell population in lysed single cell suspensions of peripheral blood of WT 1:0 (n=2, black bar), KO 0:1 (n=4, red bar), WT:KO 1:1 (n=6, orange bar) transplanted mice to distinguish donor contribution using CD45.2 surface marker (Two-way ANOVA with Turkeys multiple comparisons. * p<0.05, *** p<0.001, **** p<0.0001). Data presented as mean, error bars represent ±SD.

FIGURE 18. Competitive BMT of sorted Lin-/c-Kit+/CD71low/mid/high and whole BM cells of diseased Vav1-iCre;Nsd1fl/fl mice.

(A) Gating strategy for fluorescence activated cell sorting (FACS). Lineage depleted Lin-/c-Kit+ cells were subfractionated using CD71 surface marker into low, mid and high expressing cells. (B) Representative images of sorted Lin-/c-Kit+/CD71low/mid/high cells plated into M3434 methylcellulose. Cell numbers plated are indicated in brackets. (C) Schematic depiction of experimental setup. CD45.1+ wildtype cells were mixed with CD45.2+ sorted or whole bone marrow cells of diseased Vav1- iCre;Nsd1fl/fl mice and transplanted into lethally irradiated CD45.1+ recipients. (D) Table containing information about numbers of transplanted cells of control and Vav1-iCre;Nsd1fl/fl mice and calculated constitution of transplanted material. (E) Kaplan Meier survival curve of transplanted mice: KO 0:1 (n=3, red line), WT:KO 10:1 (n=3, yellow line), WT:KO CD71low 10:1 (n=4, light blue line), WT:KO CD71mid 10:1 (n=4, middle blue line) and WT:KO CD71high 10:1 (n=2, dark blue line). Survival is shown as percentage at days after transplantation. Median survival of KO 0:1 and WT:KO 10:1 transplanted mice was 29 days, WT:KO CD71low 10:1 41 days, WT:KO CD71mid 48 days and WT:KO CD71high 180 days. (F) Spleen weight of transplanted mice in gram: KO 0:1 (n=3, red bar), WT:KO 10:1 (n=3, yellow bar), WT:KO CD71low 10:1 (n=3, light blue bar), WT:KO CD71mid 10:1 (n=3, middle blue bar) and WT:KO CD71high 10:1 (n=1, dark blue bar) groups. Dashed line indicates average spleen weight in control whole bone marrow transplanted mice from previous experiment. (G) Peripheral blood counts of white blood cells, (H) red blood cells, (I) platelets, (J) reticulocytes (K) lymphocytes in KO 0:1 (red bar), WT:KO 10:1 (yellow bar), WT:KO CD71low 10:1 (light blue bar), WT:KO CD71mid 10:1 (middle blue bar) and WT:KO CD71high 10:1 (dark blue bar) groups (n=3/group). Dashed lines indicate average values in control whole bone marrow transplanted mice from previous experiment. Data presented as mean, error bars represent ±SD.

4.3.6 Loss of Nsd1 leads to early accumulation of erythroid