Constitutively active CaMKII Drives B lineage acute lymphoblastic leukemia/lymphoma in tp53 mutant zebrafish

Acute lymphoblastic leukemia/lymphoma (ALL) is the most common pediatric cancer and is a malignancy of T or B lineage lymphoblasts. Dysregulation of intracellular Ca2+ levels has been observed in patients with ALL, leading to improper activation of downstream signaling. Here we describe a new zebrafish model of B ALL, generated by expressing human constitutively active CaMKII (CA-CaMKII) in tp53 mutant lymphocytes. In this model, B cell hyperplasia in the kidney marrow and spleen progresses to overt leukemia/lymphoma, with only 29% of zebrafish surviving the first year of life. Leukemic fish have reduced productive genomic VDJ recombination in addition to reduced expression and improper splicing of ikaros1, a gene often deleted or mutated in patients with B ALL. Inhibiting CaMKII in human pre-B ALL cells induced cell death, further supporting a role for CaMKII in leukemogenesis. This research provides novel insight into the role of Ca2+-directed signaling in lymphoid malignancy and will be useful in understanding disease development and progression.


Introduction
Acute lymphoblastic leukemia/lymphoma (ALL) is the most common pediatric cancer, representing approximately 3,000 new cases annually in patients under the age of 20 in the United States [1].ALL originates from lymphoid progenitors in the bone marrow, with approximately 85% of pediatric lymphoid malignancies diagnosed as B lineage and 15% T lineage.While existing treatments are effective in eliminating primary disease, therapy-related complications can be severe and relapse with significantly worsened prognosis is common [2].These concerns point to the need for additional models of ALL to better understand disease etiology, progression, relapse potential, and as a platform for identification of novel therapeutic approaches.
In this study, we generated a transgenic zebrafish line that expresses a phosphomimetic, constitutively active mutant (T287D) of human CaMKII (CA-CaMKII) in lymphocytes.As previously observed in mice [22,23], CA-CaMKII on its own did not induce malignancy in zebrafish; however, expression of CA-CaMKII in tp53 mutant fish (M214K) led to an ALL phenotype.Molecular characterization of kidney marrow lymphoblasts indicated that transformation occurs in immature B cells with splenomegaly in a subset of animals.Furthermore, rag2:EGFP-CA-CaMKII; tp53 mutant lymphoblasts had reduced expression and incorrect splicing of ikaros1 (ikzf1), a gene commonly mutated or deleted in patients with B ALL [2,30,31], leading to altered B cell development.Finally, treatment of human pre-B ALL cells with a CaMKII inhibitor resulted in cell death, further supporting a role for CaMKII in human leukemogenesis.This new zebrafish model of B ALL provides insight into the role of Ca 2+ signaling in B lineage leukemia and provides additional evidence that dysregulation of CaMKII can promote disease progression.
Outside the thymus, rag2 + lymphocytes are found in the kidney marrow, the site of definitive hematopoiesis, the spleen, gut, testes, ovary, and blood of adult zebrafish [35][36][37].EGFP immunostaining of isolated kidney marrow and spleen cells revealed nuclear and cytosolic CaMKII (Fig 1F -1K).Given that the transgenic CaMKII peptide lacks a nuclear localization sequence, observed nuclear localization is likely due to hetero-oligomerization with endogenous nuclear CaMKII isoforms [32].In support of this possibility, RT-PCR profiling of wild type kidney marrow lymphocytes revealed endogenous expression of both cytosolic (δ2-E; β1-C) and nuclear (β1-K; γ2-K) CaMKII isoforms [13,14].Therefore, our stable line expresses CA-CaMKII in both the cytosol and nucleus of lymphocytes in the thymus, kidney marrow, and spleen, raising the possibility of a nuclear role for CaMKII.

Rag2:EGFP-CA-CaMKII is expressed in T cells in the thymus and immature B cells in the kidney marrow
To further confirm the tissue-specificity of our transgenic line, rag2:EGFP-CA-CaMKII fish were crossed with rag2:DsRed fish, which shows DsRed expression in T and B cells [38,39].At This result was consistent with the rag2:GFP transgenic line, where GFP positive cells were not reported on the skin [4,8].It is likely the stability of DsRed, due to the tetrameric structure, enabled the persistence of fluorescence in mature B cells, while EGFP-CA-CaMKII was degraded [40].Taken together, this suggested that EGFP-CA-CaMKII was expressed in thymic T cells but was not expressed in mature B lymphocytes on the skin [41,42].
To evaluate the extent of transgene expression in kidney marrow, we analyzed the major hematopoietic lineages by light scatter [5] and were able to define erythrocyte, myeloid, precursor and lymphocyte populations from rag2:EGFP-CA-CaMKII; rag2:DsRed fish (Fig 2G).DsRed + and EGFP + populations were found almost exclusively in the lymphoid gate, as expected (Fig 2H).Further analysis of fluorophore expression revealed the presence of EGFP single positive (gSP), DsRed single positive (rSP) and EGFP; DsRed double-positive (DP), and double-negative (DN) fractions (Fig 2I ), suggesting the possibility that EGFP-CA-CaMKII was expressed in a specific lymphocyte population.
To determine the significance of these fractions, we sorted and analyzed them for the expression of lymphoid markers at 6 mpf (Fig 2J and 2K) and 12 mpf (Fig 2L and 2M).We examined markers of B cell (rag1, rag2, igiv1s1, ighm-C, ight-C, cd79a, igic1s1) and T cell (lck) development.B cells display ordered gene expression indicative of maturation stage [43].Based on expression of selected marker genes, the most immature population was the gSP (EGFP + ) population, which expressed rag genes did not express or weakly expressed cd79a, and expressed low levels of immunoglobulin heavy (ighm, ight) and light chain (igiv1s1, igic1s1) genes.By the time B cells reached the DP (EGFP + DsRed + ) stage, they strongly expressed rag genes.They then matured to the rSP (DsRed + ) stage where rag2 expression was reduced and rag1 was absent.At the DN (EGFP -DsRed -) stage, no rag1 was detectable and rag2 was barely visible.The DN fraction also contained lck + cells, which were likely T cells, although recent published data demonstrates weak lck expression in some B cells [7].The generation of separate fluorescent gated populations was unexpected given both DsRed and EGFP-CA-CaMKII were expressed using the rag2 promoter.The presence of the gSP fraction was likely due to the more rapid maturation of EGFP compared to DsRed, [40] which accounted for the small percentage of gSP cells identified in the kidney marrow (0.10-0.54%).Furthermore, the presence of rSP cells in the kidney marrow, similar to DsRed + cells on the skin, suggested EGFP-CA-CaMKII was degraded as cells matured, while DsRed persisted due to the stability of the tetramer [40].The differential gene expression observed at 6 and 12 mpf could be due to alterations in B cell generation and differentiation associated with aging, similar to humans [44].Thus, the most immature B cells, due to expression of rag1 [43], were found in the gSP and DP population, while the most mature B cells were found in the rSP and DN fractions.Therefore, we concluded that EGFP-CA-CaMKII is expressed in immature B cells.

Lymphocyte hyperplasia in rag2:EGFP-CA-CaMKII; tp53 mutants
Rag2:EGFP-CA-CaMKII fish were phenotypically normal and displayed no significant health issues, similar to mice expressing CA-CaMKII in thymocytes [22].Although leukemia/lymphoma patients often display increased CaMKII activity [21,24,25], constitutive activation of CaMKII alone does not appear to drive leukemic transformation, suggesting that additional genetic alterations are required.
Inactivating mutations in the tumor suppressor TP53 occur in approximately 5-16% of ALL patients [45][46][47][48][49].Although discovery mutations in TP53 often occur in conjunction with a low hypodiploid karyotype, 10-20% of relapse ALL patients have somatic mutations in TP53 without this subtype [48,50].These TP53 alterations lead to reduced function and are linked with treatment failure [46][47][48]50] identifying the need for novel targets for therapeutic intervention.TP53 is activated in response to DNA damage, and is a key mediator of tumor suppression responses by pathways including ARF (CDKN2A), leading to cell cycle arrest or apoptosis.Mutations and deletions in ARF occur frequently in ALL [51], inhibiting TP53dependent apoptosis.A zebrafish ortholog of ARF has not been identified [52], but tp53 mutant zebrafish have been used to study interruption of the ARF-dependent signaling pathway as well as directly study tp53 function in leukemic transformation [52].The established zebrafish tp53 mutant line carries a point mutation in the region encoding the DNA binding domain (M214K), leading to transcriptional inhibition of target genes.Homozygous tp53 mutants are viable, with less than 5% of animals spontaneously developing malignancies by one year of age.Tumor incidence was estimated at 28% by 16 months, with the majority of animals developing malignant peripheral nerve sheath tumors (MPNST), but not leukemia [53].Rag2:EGFP-CA-CaMKII fish were crossed into the tp53 mutant background to examine their combinatorial potential to drive leukemic transformation.
Leukemia in humans is diagnosed when lymphoblasts exceed 25% of the cell population in the bone marrow or peripheral blood, in accordance with the National Comprehensive Cancer Network guidelines [54].The elevated lymphocyte counts observed in some individual zebrafish exceeded the threshold for diagnosis of leukemia (Fig 3).Since initial FACS analyses were conducted on fish prior to overt signs of illness, we prepared kidney marrow (Fig 4A -4C) and spleen smears from adult zebrafish that appeared ill, as demonstrated by lethargy and failure to eat.The proportion of erythrocytes, lymphocytes, and myelomonocytes in the kidney marrow (Fig 4D ) and spleen (Fig 4E) was unchanged in fish carrying the rag2:EGFP-CA-CaMKII transgene on a wild type or tp53 heterozygous background.However, the percentage of erythrocytes was significantly decreased and the percentage of lymphocytes more than doubled in the kidney marrow and spleen of rag2:EGFP-CA-CaMKII; tp53 mutants.(Fig 4D and 4E).Thus, rag2: EGFP-CA-CaMKII; tp53 mutants progress from lymphoid hyperplasia to overt malignancy.

ALL (Fig 5U)
. Overt illness occurs rapidly, where fish with leukemia/lymphoma were unable to eat and succumb to illness within four-to-six hours, resulting in 35% of fish dying prior to analysis.However, leukemia developed solely in fish that expressed lymphoid targeted EGFP-CA-CaMKII in tp53 mutants, further suggesting inappropriate activation of CaMKII is a second hit leading to leukemic transformation.
To further assess B cell development, kidney marrow lymphocytes were analyzed for genomic rearrangement of the variable (V), diversity (D), and joining (J) gene segments of the immunoglobulin heavy chain locus in rag2:DsRed, rag2:EGFP-CA-CaMKII, tp53 mutant, and rag2:EGFP-CA-CaMKII; tp53 mutant fish (Fig 6C).The igh locus is comprised of a cluster of V gene segments followed by DJ constant (C) gene segments for igt and then DJC gene segments for igm [55][56][57].In order for igm rearrangement to occur, the intervening igt DJC sequence is deleted (Fig 6B) [56].Therefore, kidney marrow lymphocytes were assayed for productive rearrangements of igt and igm from variable domains 1, 2, and 3 to (DJ) T or (DJ) M by RT-PCR (Fig 6B).Igm VDJ-rearrangement was observed in lymphocytes of all fish examined (Fig 6C ), however rag2:EGFP-CA-CaMKII fish had reduced productive IgVH3-Jm rearrangement, while tp53 mutant fish had reduced IgVH1-Jm rearrangement and rag2:EGFP-CA-CaMKII; tp53 mutant fish had reduced productive IgM rearrangement from all three V domains analyzed (Fig 6D).Unlike igm, igt rearrangements were not identified in all fish (Fig 6C, red arrows), where rag2:EGFP-CA-CaMKII fish failed to undergo V to DJ rearrangement in all samples analyzed (Fig 6C).In contrast, tp53 mutant fish displayed productive rearrangements for all samples, while rag2:EGFP-CA-CaMKII; tp53 mutant fish had reduced productive rearrangements for IgVH1-Jt and IgVH3-Jt but normal IgvH2-Jt rearrangements (Fig 6E).Our findings point to two main conclusions: First, rag2:EGFP-CA-CaMKII fish rearranged igm loci in preference to igt on a wild type background.Second, productive rearrangements are reduced in both the igt and igm loci of rag2:EGFP-CA-CaMKII;tp53 mutant fish.

Pharmacological inhibition of CaMKII alters cell cycle distribution and induces cell death in pre-B ALL cells
Increased expression and activation of CaMKII has been observed in both cell lines and patient samples of diverse hematological malignancies [21,24,[64][65][66].Specifically, it was reported that NALM6 cells, a human pre-B ALL cell line, expressed elevated levels of γ CaMKII compared to normal blood as well as high levels of autophosphorylated CaMKII [28,29].RT-PCR analysis of CaMKII identified expression of cytosolic variants of CaMK2G (γ-B, γ-C, γ-E, γ-H) and CaMK2D (δ-E) in NALM6 cells.Therefore, NALM6 cells were treated with KN-93, an established CaMKII pharmacological inhibitor, to determine if CaMKII is functionally important in pre-B ALL maturation [17,67,68].Cells were treated with 2.5μM, 5μM, or 10μM KN-93 and analyzed after HEMA3 staining (Fig 8A -8D).Cell numbers were reduced in KN-93 treated samples and chromatin appeared more condensed (Fig 8D).Time course analysis confirmed cell number occurs in a dose dependent manner beginning at day 2 and continuing through day 3 (Fig 8E).Analysis of cell cycle distribution noted an increase in S phase and a reduction in G1 and G2/M phase distribution with 5μM treatment, and an increase in sub G1 and S phase distribution and reduction in G2/M distribution with 10μM treatment (Fig 8F).Our results indicate that CaMKII inhibition alters cell-cycle distribution and likely induces cell  ).https://doi.org/10.1371/journal.pgen.1011102.g007death in a pre-B ALL cell line.Analysis of early and late stage apoptosis using PI and Annexin V (Fig 8G and 8H) indicated that KN-93 did not affect early apoptosis, but significantly promoted late apoptosis (Fig 8H).These results support a role for CaMKII in the proliferation of human pre-B ALL cells in culture.

Discussion
ALL is the most common childhood cancer and comprises approximately 25% of all pediatric cancers.ALL is most commonly identified in B lineage cells and is diagnosed when lymphoblast composition of the bone marrow or peripheral blood exceeds 25% [54].Extramedullary involvement with splenomegaly or hepatomegaly occurs in approximately 20% of patients [54].Our results demonstrate that activated CaMKII on a tp53 mutant background in zebrafish induces B cell hyperplasia in both the kidney and spleen progressing to an overt leukemia/ lymphoma phenotype as early as four months of age, with only 29% of fish surviving the first Dysregulation of Ca 2+ signaling is associated with multiple human disorders including, heart failure, Alzheimer's, and cancer development in both animal models and patients [11,69,70].Ca 2+ is normally transiently elevated to activate downstream signaling molecules, such as CaMKII.Prolonged activation of CaMKII has been linked to diverse malignancies, including leukemia [21,71,72].Patient samples and cells in culture show increased CaMKII expression as well as increased autophosphorylation of CaMKII at T287, leading to persistent Ca 2+ -independent activity [21,29].In addition, the tumor suppressor protein phosphatase 2A (PP2A) is often inactivated in hematopoietic malignancies [73][74][75][76].Since, PP2A dephosphorylates CaMKII at T287, returning the holoenzyme to its Ca 2+ -dependent regulatory state [77], inappropriate activation of CaMKII would be an expected result of loss, mutation, or inactivation of PP2A.Furthermore, increased CaMKII activation could result from dysregulation of cytosolic Ca 2+ concentrations.In leukemia, decreased expression of SERCA3 or increased expression of IP3R2, inhibits maintenance of internal ER Ca 2+ stores leading to activation of Ca 2+ -dependent signaling molecules [11].
Although expression of CA-CaMKII did not induce leukemia development on its own, increased CaMKII activity was associated with new progression to hematologic malignancy on a tp53 predisposition background, indicating it functions as an additional "hit" during transformation.Consistent with this role, multiple leukemic cell lines display increased CaMKII expression and autophosphorylation in the presence of background leukemia drivers, such as BCR-ABL [21,78].In addition, expression of camk2d1 is increased in immature B lymphoblasts but not T cells of zebrafish with cmyc-driven T, B, and mixed lineage leukemia [7].Furthermore, increased expression of CaMKII was identified in a subset of pediatric patients with B ALL [79,80].Altogether, these results suggest altered CaMKII expression promotes leukemia transformation on different genetic backgrounds, but is not the primary driver of malignancy.Mutations and deletions in tumor suppressors, such as TP53 and ARF, have been linked to ALL transformation [47,51].Although TP53 mutations infrequently occur during the discovery phase of ALL, inactivating TP53 mutations are common upon relapse [47].In addition, deletions in ARF are commonly identified in patients with ALL, with an incidence rate varying from 18-45% [51,81].ARF signals to TP53 to enable cell cycle arrest or apoptosis after DNA damage [82].Although an ortholog of ARF has not been identified in zebrafish, TP53 is a key mediator of ARF tumor suppression, and inhibition of the TP53 cellular response likely mimics ARF deletion, as previously reported [52].
Expression of EGFP-CA-CaMKII in lymphocytes of tp53 mutant zebrafish caused lymphoblast proliferation, similar to ALL.Molecular analysis of leukemic lymphoblasts confirmed B lineage with expression of rag1, rag2, cd79a, igiv1s1, igic1s1, pax5, ighm-C, and ight-C and lack of T cell markers lck and tcrd-C.Immaturity of tumor cells was indicated by blast morphology and expression of rag1, which is not expressed in mature, quiescent B cells [43].Further analysis of EGFP+ leukemic lymphoblasts determined that the expression of the zinc-finger transcription factor, ikzf1, was reduced and incorrect splicing induced nonfunctional variants.Ikaros1 (Ikzf1) is an essential gene during lymphoid differentiation and is often deleted or mutated in patients with B ALL [2,30,31].In zebrafish, mutations in ikzf1 leads to lower thymopoiesis efficiency [57,83], loss of kidney marrow B cell igt rearrangements and reduced productive igm rearrangements [57].Zebrafish ikzf1 mutants do not develop overt leukemia or lymphoma, likely requiring an additional genetic mutation or alteration [57,83].Similar to ikzf1 mutants, rag2:EGFP-CA-CaMKII; tp53 mutant B cells had reduced productively rearranged igm and igt alleles.Genomic VDJ recombination is essential for maturation of the adaptive immune system.Without productive rearrangements, the immune system is compromised which could lead to chromosomal rearrangements and B cell malignancies [84].The limited number of productive IgH rearrangements in rag2:EGFP-CA-CaMKII; tp53 mutant fish could reduce the effectiveness of the adaptive immune system.Leukemic fish could therefore be susceptible to infections, leading to lethargy and death.Thus, reduced expression and frameshift mutations of ikzf1 in rag2:EGFP-CaMKII; tp53 mutant kidney marrow B lymphoblasts could lead to reduced productive VDJ recombination and thus inhibit B cell maturation, promoting leukemia.
Although rag2-driven oncogenes in previous zebrafish models predominantly generated T-ALL [7,8], our model did not express T cell lineage markers.The specific effect on B cells is likely due to the combinatorial effect of increased activation of CaMKII and the tp53 mutant background.This is the first study to show a role for activated CaMKII in B ALL using a model system and the second zebrafish B ALL model driven by rag2 [7].Therefore, this model provides novel insight into the role of calcium signaling in leukemia maturation and can be used to better understand the role of CaMKII in B ALL.

Ethics statement
All animal research was conducted under approved IACUC protocols, and in compliance with the Institutional Animal Care and Use Committee (IACUC) of Virginia Commonwealth University and according to the American Veterinary Medical Association (AVMA) guidelines.

Transgenic line generation
Gateway cloning (Invitrogen) was used to produce a destination transgenesis construct with the zebrafish recombination activating gene 2 (rag2) promoter [8] driving expression of a constitutively active (CA, T287D) CaMKII [13,16], flanked by Tol2 transposase cis targeting elements [3]. 25 ng of rag2:EGFP-caHsa.CAMK2_T287D was co-injected with 25 ng of Tol2 transposase mRNA into AB embryos at the one-cell stage.P0 fish were raised and outcrossed to generate stable F1 Tg(rag2:EGFP-caHsa.CAMK2_T287D) animals (referred to as rag2: EGFP-CA-CaMKII in the text).F2 stable transgenics were crossed into the tp53 mutant background to generate a stable line carrying both transgene and mutant alleles.Transgene copy number was not determined in rag2:EGFP-CA-CaMKII; tp53 wild type or mutant fish since EGFP brightness was not discernible between heterozygote and homozygote fish.The functionality of the T287D mutation was previously demonstrated in both cells in culture and zebrafish embryos [13,16,33].

Flow cytometry
Zebrafish kidneys and spleens were dissected into 1X PBS + 4% FBS, passed through a 40 μm filter, centrifuged at 800 rpm for 5 minutes, and washed twice in 1X PBS + 4% FBS [8].Cell populations were incubated with DAPI to eliminate dead cells and analyzed on a Fortessa (Becton-Dickinson, B-D) analyzer or sorted on a FACS Aria II or Fusion Aria high-speed analyzer/sorter (B-D) and saved in either TRIzol reagent (Invitrogen) or 1X PBS + 4% FBS.

Fig 8 .
Fig 8. Pharmacological inhibition of CaMKII induces cell death in human pre-B ALL cells.HEMA3 stained NALM6 cells treated with 2.5μM, 5μM, and 10μM KN-93 at 48h (A-D).NALM6 growth curves were assessed at 24-hour intervals until 72h after KN-93 treatment.(E, n = 3).Cell cycle distribution was assessed after PI staining in control, 5μM, and 10μM KN-93 treated NALM6 cells at 48h (F, n = 5).NALM6 cells were stained with Annexin V and PI after KN-93 treatment and analyzed using flow cytometry at 48h.The lower left quadrant are cells that are negative for Annexin V and PI, the upper left quadrant is PI positive only indicative of necrosis, the upper right quadrant identifies cells that are positive for both Annexin V and PI indicating late apoptosis, and the bottom right quadrant shows cells that are Annexin V positive, which indicates early stages of apoptosis (G).The bar graph shows the percent of cells that are in early and late stage of apoptosis from four experiments (H).P values were calculated using one-way ANOVA followed by Tukey HSD.* p<0.05 and ** P<0.01.Scale bar: 20 μm in A. https://doi.org/10.1371/journal.pgen.1011102.g008