Page 241 - 2021_05-Haematologica-web

Letters to the Editor
The multifaceted role of protein kinase CK2 in
high-risk acute lymphoblastic leukemia
Acute lymphoblastic leukemia (ALL) is an aggressive malignancy of developing lymphocytes. Despite out- standing overall cure rates, patients with the refractory or relapsed disease have a poor prognosis.1 In order to improve treatments for these high-risk (HR)-ALL patients, it is critical to gain an in-depth understanding of the disease pathogenesis. The enhanced expression of the protein kinase CK2 gene and proto-oncogene MYC are common in T cell ALL (T-ALL) and B cell ALL (B-ALL).2-6 CK2 is a constitutively active serine/threonine kinase composed of two catalytic (α or α') and two regu- latory (b) subunits that are overexpressed in a broad spec- trum of human cancers.7 Despite the demonstrated anti- leukemic efficacy of CK2 inhibitors,8 how CK2 con- tributes to HR-ALL development remains incompletely understood. Here we utilized transgenic zebrafish mod- els to elaborate the multifaceted role of CK2 in HR-ALL pathogenesis, providing therapeutic implications for this stubborn disease.
for tumor onset using previously defined criteria.12,13 At 4 weeks of age, all groups showed normal-sized thymi. However, by 6 weeks, all three fish lines exhibited evi- dence of tumor initiation compared to Tg(lck:EGFP) and Tg(rag2:mCherry) controls (Figure 1C; Online Supplementary Figure S1B). By 12 weeks of life, tumors developed in more than 90% of Tg(rag2:MYC- ER;lck:EGFP;lck:CK2αwt;rag2:mCherry) fish, referred to as MYC-ER;CK2αwt (Figure 1B and C). However, tumors developed in less than 60% of Tg(rag2:MYC- ER;lck:EGFP) fish, referred to as MYC-ER (Figure 1B and C).
Overexpression of the CK2α subunit under the
Next, we questioned whether CK2α could hasten the progression of MYC-induced ALL by quantifying the tumor burden in the above three groups of fish. We found that ALL developed in MYC-ER;CK2αwt fish much more aggressively, as demonstrated by a significantly heavier tumor burden in these fish compared to those in MYC-ER sibling fish (Online Supplementary Figure S2A and B). However, overexpression of CK2αK68M failed to enhance disease aggression as the tumor burden in MYC- ER;CK2αK68M fish was similar to those in MYC-ER fish (data not shown). Since MYC-ER fish develop both T- and B-ALL,2,11 we then asked which types of leukemia MYC-ER;CK2αwt fish developed by performing semi- quantitative reverse transcriptase polymerase chain reac- tion (RT-PCR) using zebrafish T- and B-cell specific primers.14 Our results show that MYC-ER;CK2αwt fish also developed ALL of T and B lineages (Online Supplementary Figure S3).
immunoglobulin gene promoter induces9 low penetrance
of T-cell lymphomas in a murine model. In order to fur-
ther understand the oncogenic potential of CK2 in T and
B lineages, we generated transgenic zebrafish that over-
express the wild-type or the kinase-dead version (K68M)
of the human CK2α gene in T and B cells through the 2,10
zebrafish tyrosine kinase gene (lck) promoter. Western blotting analysis revealed elevated expression of CK2α in transgenic CK2 fish, compared to age-matched wild-type (wt) fish (Online Supplementary Figure S1A). Despite rela- tively normal thymus development and no observable difference in fish survival (Online Supplementary Figure S1B), lymphocytes in Tg(lck:CK2wt;rag2:mCherry) fish survived much longer than the control Tg(lck:EGFP) or Tg(rag2:mCherry) transgenic fish (Online Supplementary Figure S1C). By 8 months, CK2 transgenic fish still had clearly defined red-fluorescent thymi, while control transgenic fish began to lose their thymic fluorescence as early as 5 months of age (Online Supplementary Figure S1C and data not shown). Strikingly, CK2 transgenic fish can retain their thymic fluorescence till 18 months. In order to determine the effect of CK2α in inducing lymphoid malignancies in zebrafish, starting at 21 days post-fertil- ization (dpf), we monitored both wt and mutant CK2 transgenic fish at least once a month until 2 years of age and found no tumor development in these fish (Online Supplementary Figure S1C and data not shown). Additionally, we also overexpressed CK2α under the zebrafish rag2 promoter and also failed to observe tumor development in this fish line. These results indicate that CK2 overexpression alone has very limited oncogenic potentials.
Despite the early knowledge that CK2 accelerates MYC-induced T-ALL,9 several questions remain: i) can CK2 and MYC synergize to promote B-ALL?, ii) does CK2’s tumor-promoting effect solely depend on its enzy- matic activity? and iii) how does CK2 contribute to dif- ferent stages of ALL development? To this end, we bred our lck-promoter-driven wt or kinase-dead CK2 trans- genic fish to conditional Tg(rag2:MYC-ER;lck:EGFP) fish, in which aberrant MYC activity is regulated by tamoxifen and induces leukemia in both T and B lineages.2,11 We raised their offspring in fish water containing 4-hydroxy- tamoxifen (4HT) beginning at 5 dpf when thymic fluores- cence was first visible (Figure 1A), and monitored the fish
In order to determine whether MYC-induced transfor- mation is restricted to the particular stages of lymphocyte development, we treated MYC-ER fish with 4HT at 30 dpf instead of 5 dpf, and monitored fish for tumor devel- opment with weekly fluorescent imaging (Figure 2A). Surprisingly, none of these MYC-ER fish developed tumors after 8 weeks of 4HT treatment (Figure 2B and C). However, if these fish were treated with 4HT at 5 dpf, more than 30% of MYC-ER fish had already developed tumors at this time (Figure 1C). Next, we determined if the enhanced CK2α expression could overcome this tem- poral restriction of lymphocyte transformation. In order to do so, we bred CK2α transgenic fish to MYC-ER fish and treated the fish with 4HT at 30 dpf. Strikingly, tumors started to arise in the MYC-ER;CK2αwt fish with- in 1 week of 4HT treatment (Figure 2C). Within less than 2 weeks of 4HT treatment, approximately 80% of MYC- ER;CK2αwt fish developed aggressive ALL (Figure 2B and C). These results demonstrate that CK2 can overcome the temporal restriction of MYC-mediated lymphocyte transformation and induce ALL at a later developmental stage.
Since the aggressive nature of leukemia in MYC- ER;CK2αwt fish depends on the kinase activity of CK2 (Figure 1), we next performed phos-tag western blotting to determine whether enforced CK2 expression increases MYC phosphorylation in vivo. Compared to tumors in MYC-ER fish, we detected increased CK2α and relatively more phosphorylated MYC (upper bands) protein levels
Interestingly, overexpressing the enzyme-dead version of CK2α in Tg(rag2:MYC-ER;lck:EGFP;lck:CK2αK68M; rag2:mCherry) fish, referred to as MYC-ER;CK2αK68M, failed to accelerate the disease, with approximately 50% of fish developing tumors at 12 weeks of life (Figure 1B and C). These results demonstrate that the HR-ALL development depends on the enzymatic activity of CK2 since wt, but not kinase-dead CK2α, significantly accel- erated the onset of MYC-induced ALL.
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