Abstract
Precursor-B cell acute lymphoblastic leukemia (pre-B ALL) is the most common pediatric cancer, but there are no useful zebrafish pre-B ALL models. We describe the first highly-penetrant zebrafish pre-B ALL, driven by human MYC. Leukemias express B lymphoblast-specific genes and are distinct from T cell ALL (T-ALL)—which these fish also develop. Zebrafish pre-B ALL shares in vivo features and expression profiles with human pre-B ALL, and these profiles differ from zebrafish T-ALL or normal B and T cells. These animals also exhibit aberrant lymphocyte development. As the only robust zebrafish pre-B ALL model and only example where T-ALL also develops, this model can reveal differences between MYC-driven pre-B vs. T-ALL and be exploited to discover novel pre-B ALL therapies.
Statement of significance We describe the first robust zebrafish pre-B ALL model in MYC-transgenic animals known to develop T-ALL, revealing the only animal model with both human ALL types. We also describe aberrant multi-lineage lymphopoiesis. This powerful system can be used to study MYC-driven leukemogenesis and discover new pre-B ALL targeted therapies.
Introduction
Acute lymphocytic leukemia (ALL), a common cancer and the most prevalent childhood malignancy, comprises >25% of pediatric neoplasia in the U.S., with ∼85% being pre-B ALL (1, 2). Relapses are all-too-common, making ALL the highest cause of pediatric cancer-related death (3). Thus, there is a dire need for animal models of pre-B ALL to identify new molecular targets and discover new therapies, but efforts are impeded by a lack of in vivo models amenable to genetic and drug screens.
Zebrafish (Danio rerio) provide one potential solution, since they can model human leukemias accurately (4), have practical advantages (genetic tractability, high-throughput screens, cost), and share hematopoietic, oncogenic, and tumor suppressive pathways with humans (5). These features permitted the creation of several zebrafish T cell ALL (T-ALL) models that mimic the human disease (6-10), which subsequently led to key findings in T-ALL genetics, disease progression mechanisms, and signaling (11-16), as well as facilitating screens for new treatment agents (17, 18). However, despite the even greater clinical impact of pre-B ALL, effective zebrafish models lag behind. A single report of zebrafish B-ALL using transgenic ETV6-RUNX1 (19) had low penetrance and long latency (∼3% by 1 year), and no subsequent reports with this or any other B-ALL model exist.
Here we utilized a cell-specific fluorophore, lck:eGFP (20), that labels zebrafish B and T cells differentially to discover the first robust D. rerio B-ALL model. Surprisingly, B-ALLs occur in an already-established T-ALL model driven by transgenic human MYC (10), and they are so prevalent that many animals actually have coincident B-and T-ALL. An intensive investigation of this new model using several approaches revealed a number of important findings. First, hMYC-induced B-ALL are pre-B subtype, express immature B cell transcripts, and like human pre-B ALL, spread aggressively to lymphoid and non-lymphoid tissues. Second, pre-B ALL express low levels of lck, and thus are dimly-fluorescent in these animals, unlike the brightly-fluorescent T-ALL of this model. Low LCK expression is conserved in human pre-B ALL. Third, in addition to their differential lck:eGFP expression, we report a two-gene classifier that distinguishes pre-B from T-ALL in hMYC fish. Finally, expression profiles of zebrafish pre-B ALL, T-ALL, and normal B and T cells revealed abnormal lymphopoiesis that may underlie the molecular pathogenesis of hMYC-driven ALL. In summary, we report a novel and robust pre-B ALL model, the first in zebrafish. Besides its value for genetic and drug screens, to our knowledge, hMYC fish represent the only animal model that develops both pre-B and T-ALL, providing a unique tool to explore molecular mechanisms of both human ALL types in the same genetic context, or even the same animal.
Results
Human MYC induces two zebrafish ALL types with distinct expression signatures
Mammalian Myc/MYC transgenes driven by a D. rerio rag2 promoter induce zebrafish T-ALL (6, 10). To detect and monitor ALL progression, we built double-transgenic fish by crossing Tg(rag2:hMYC) to Tg(lck:eGFP) fish, where a zebrafish lck promoter controls GFP expression (20). Henceforth, we refer to this double-transgenic line as hMYC;GFP. To study T-ALL in this system, we performed RNA microarray on FACS-purified GFP+ ALL cells, analyzing 10 hMYC;GFP malignancies and 3 cancers from hlk fish (9), another zebrafish T-ALL model (see Fig. 1A for example animals).
Unsupervised analysis divided hlk and hMYC;GFP malignancies precisely, emphasizing fundamental differences in ALL from different D. rerio models (Fig. 1A). Unexpectedly, hMYC;GFP ALL also clustered into two subgroups with distinct gene expression profiles (GEPs). To further investigate these groups, we used hlk T-ALL as a reference and designated the 4 ALL closest to hlk as hMYC-1, and the 6 ALL at the far right as hMYC-2 (blue and orange samples in Fig. 1A).
Separate comparisons of hMYC-1 or hMYC-2 vs. hlk ALL revealed that B cell-specific genes were up-regulated by both types of hMYC;GFP ALL (pax5, btk, cd81, etc.; Fig. 1B), with hMYC-2 ALL over-expressing additional B cell-specific genes (syk, blnk, ighm). Ingenuity Pathway Analysis™ (IPA) of these differentially-expressed genes showed enrichment and activation of “PI3K-Signaling in B-Lymphocytes”, “B-cell receptor-signaling” and “FcγRIIB-signaling in B-cells” pathways by hMYC-2 ALL, but not hMYC-1, relative to hlk T-ALL (data not shown). To further investigate the unanticipated expression of B cell genes by hMYC ALL, we repeated unsupervised analysis using only 14 statistically-significant B cell-specific genes. Remarkably, this signature classified hlk vs. hMYC ALL perfectly and largely reformed both the hMYC-1 and hMYC-2 subclasses (Fig. 1C).
Expression of B cell genes by hMYC cancers was unexpected, because B-ALL has never been described by several laboratories—including ours—that study transgenic Myc/MYC zebrafish (6, 10, 11, 15, 18, 21). Yet microarrays clearly demonstrated B cell genes (ighm, blnk, pax5) were expressed at high, medium, and low levels by hMYC-2, hMYC-1, and hlk ALL, respectively, with T cell-specific itk showing the opposite pattern (Fig. 1D). We hypothesized that hMYC-1 and -2 cancers might contain different proportions of lymphocytes, with hlk being “pure” T-ALL, but hMYC ALL comprised of varying amounts of T-ALL, B-ALL, and/or B cells. Alternatively, leukemias can express aberrant markers (22), and hMYC might de-differentiate ALL, obscuring cell identities. In either case, B cell genes were high in hMYC-2 and detectable in hMYC-1 also, so we next sought to definitively identify the cellular composition of hMYC cancers.
B-ALL and T-ALL each occur in hMYC;GFP animals, with different GFP intensities
To definitively identify hMYC;GFP ALL as they first developed, we used serial fluorescent microscopy to monitor unaffected animals (i.e., fish lacking visible cancers). In young adults (3-6 months), we observed two phenotypes: brightly-fluorescent cancers originating in thymus and dimly-fluorescent cancers with variable thymic involvement (Figs. 1E, S1). To distinguish these, we used “low-exposure” settings that detected only bright cancers [Figs. 1E(a) vs. (c), S1A vs. S1B], and “high-exposure” settings that revealed dim ALL which were otherwise not visible [Figs. 1E(c) vs. (f), S1B vs. S1D]. Dim ALL differed from non-cancerous hMYC and lck:eGFP control animals that showed only normal thymic fluorescence (Fig. 1F).
Flow cytometric analyses confirmed microscopy findings, with bright and dim ALL showing distinct, ≥10-fold GFP intensity differences [Fig. 1E(g-i)]. Thus, we could discern ALL with only bright (GFPhi), only dim (GFPlo), or both cell populations. We analyzed ALL from 27 hMYC fish with fluorescent cancers at 6 months of age and found 7 dim ALL with near-exclusively GFPlo cells (Fig. 2A) and 14 GFPhi-only ALL (Fig. 2C). Intriguingly, we also found 6 mixed-ALL that contained distinct populations of both GFPhi and GFPlo cells (Fig. 2B). Remarkably, these 27 animals developed 33 total ALL, 13 GFPlo and 20 GFPhi.
We next tested FACS-purified GFP+ dim, bright, or mixed-ALL cells for B cell-(pax5, cd79b, ighz, etc.), T cell-(cd4, cd8, il7r, etc.), and lymphoblast-(rag2, igic1s1, etc.) specific transcripts, as well as the GFP and hMYC transgenes by quantitative reverse-transcriptase PCR (qRT-PCR), analyzing all GFP+ cells as one population without separating GFPlo and GFPhi peaks. Dim/GFPlo ALL expressed B, but not T, cell-specific genes [Fig. 3A(a-d)]. Low lck and GFP levels in dim ALL matched their weak in vivo fluorescence. Conversely, bright/GFPhi cancers expressed only T cell genes. Mixed-ALL expressed both B and T cell genes at intermediate levels. Overall, expression correlated exactly with dim/GFPlo vs. bright/GFPhi phenotypes, and only mixed-ALL, which contained GFPlo and GFPhi cells, co-expressed genes of both cell types. Based on these findings, we conclude dim/GFPlo ALL are B-lineage ALL. Mixed-ALL always exhibited distinct GFPlo and GFPhi cell populations (Fig. 2B) and expressed B-and T-lineage genes [Fig. 3A(a-d)], so we deduce mixed-ALL are not biphenotypic, but simultaneous B-and T-ALL in one animal. B-vs. T-lineage ALL could be unambiguously distinguished by igic1s1 [Fig. 3A(e)], a homologue of the IGLL1 surrogate Ig light chain gene expressed by only immature B cells (23). Only dim and mixed-ALL expressed igic1s1, but every ALL showed similar levels of the V(D)J recombination enzyme rag2 [Fig. 3A(f)]. Based on igic1s1 and rag2 results, which only immature B cells co-express, we conclude hMYC B-ALL are, in fact, pre-B ALL. A zebrafish rag2 promoter regulates hMYC, so it is logical that rag2+ B-lymphoblasts (i.e., pre-B cells) are affected, just like T-ALL in this model (6, 10). Moreover, similar hMYC levels in pre-B and T-ALL [Fig. 3A(f)] indicate this transgene has similar oncogenic potency in both lymphocyte lineages. Surprisingly, rag1 was much lower in pre-B ALL [Fig. 3A(e)], making igic1s1 and rag1 a two-gene panel that can distinguish hMYC ALL types independent of lck or GFP levels. Mammalian B-and T-lymphoblasts co-express RAG1 and RAG2, so dichotomous rag1 levels were unexpected. However, unlike mammals, rag2-mutant zebrafish lack T cells, yet retain functional B cells (24), further suggesting V(D)J recombination may differ between mammals and D. rerio.
As predicted by different in vivo fluorescence [Figs. 1E(a-f), S1] and GFPlo vs. GFPhi cytometric results [Figs. 1E(g-i), 2], lck and GFP also differed markedly between pre-B and T-ALL [Fig. 3A(d)]. A zebrafish lck promoter regulates GFP (20), and pre-B ALL expressed little lck or GFP, while T-ALL expressed both abundantly. In agreement, lck mRNA correlated with weighted median fluorescence intensity (wMFI; Fig. 3B), with each ALL type clustering separately, proving lck levels—and thus, cellular fluorescence—distinguish pre-B vs. T-ALL in this model. We also confirmed GFP mRNA and protein levels agree by Western blot (WB), with much higher amounts of total protein needed to detect GFP in pre-B ALL compared to T-ALL (Fig. 3C).
lck is generally considered to be T cell-specific (20), but zebrafish NK and myeloid cells also express lck (25, 26). Pertinent to our study, we analyzed public data from different maturation stages of human lymphocytes (27) and found pre-B cells expressed higher LCK than naïve and mature B cells, although below that of T cells (Fig. S2A). Additionally, Microarray Innovations in Leukemia (MILE) data (28) from 1,816 leukemia patients also showed LCK levels in pre-B ALL equal to many T-ALL (Fig. S2B). Thus, human and zebrafish pre-B ALL both express LCK/lck.
Zebrafish pre-B ALL resembles human pre-B ALL morphologically
To examine pre-B ALL histology, we analyzed hMYC fish with dim ALL. Hematoxylin and eosin (H&E) stains showed lymphoblast infiltration of the kidney-marrow, thymus, liver, and elsewhere (Figs. 3D, S3A). Marrow hypertrophy was often profound, with marrow expansion and invasion through muscle into subcutaneous tissue and skin (Fig. S3A, pre-B3, -B4). Yet despite these massive disease burdens, fish remained only dimly fluorescent. As in humans, pre-B and T-ALL were indistinguishable by H&E (Fig. S3A-B), and both were markedly abnormal compared to control animals (Figs. 3E, S3C).
Immunohistochemical analysis (IHC), however, could discriminate pre-B from T-ALL, with very faint anti-GFP staining in GFPlo ALL vs. strong signals in GFPhi ALL (Figs. 3F, pre-B2 vs. T-ALL1, S4A-B), and only remnant thymic tissue showing strong signals in pre-B ALL fish (Figs. 3F, pre-B2 and S4A, pre-B5, -B6). Consistent with this, regions stained weakly by anti-GFP (Ab+) corresponded to dimly-fluorescent anatomic regions (Figs. 3F, S4A; 1000X panels). Because Ab recognizing zebrafish lymphocyte proteins are not available, we used RNA in situ hybridization (ISH; RNAscope™) to independently test cell identities using probes for hMYC and B cell-specific cd79b (Fig. 4A-C). hMYC labeled pre-B ALL strongly (Fig. 4A; H&E of this animal shown in Fig. 3D), including cells in the thymus and kidney-marrow (Fig. 4B-C, pre-B1). These same areas were also cd79b-positive, confirming B-lineage. Thymi of hMYC control fish were avidly hMYC+, but had few cd79b+ cells (Fig. 4B, D, hMYC Ctrl), indicating thymic B cells are sparse unless pre-B ALL is present. Similarly, hMYC control marrow had fewer dually hMYC+/cd79b+ cells (Fig. 4C-D), with normal kidney-marrow architecture, including renal tubules. hMYC was absent in the thymus of control lck:eGFP fish (Fig. 4B, lck:eGFP) proving probe specificity, and showed rare cd79b+ B cells, demonstrating few thymic B cells in WT fish. We examined a different animal with disseminated pre-BALL and localized T-ALL, based on microscopy and IHC findings (see microscopy, IHC, and H&E in Fig. S4A, pre-B6), adding a T-cell specific probe, lat, to distinguish pre-B vs. T-ALL. RNA ISH demonstrated cells that were hMYC+/cd79b+/lat-had completely replaced the marrow and thymic cortex (Fig. 4E), with GFPhi hMYC+/cd79b-/lat+ cells remaining only in an enlarged thymic medulla (i.e., localized T-ALL). Similar results were seen in a second animal with near-complete thymic ablation by pre-B ALL (Fig. S4C; Fig. 3F, pre-B2 shows microscopy and IHC of this specimen). In summary, in vivo fluorescence, cytometric GFP intensity, qRT-PCR, WB, and RNA ISH all prove dim cancers in hMYC;GFP fish are pre-B ALL with organ distributions similar to human pre-B ALL.
Zebrafish pre-B ALL remain GFPlo/lcklo in every tissue
Pre-B ALL disseminated aggressively (Figs. 3D-F, 4, S3A, S4A, C). To test whether pre-B ALL cells retained a GFPlo/lcklo phenotype in every niche, we examined thymus, marrow, spleen, blood, and muscle and viscera by flow cytometry (Fig. 5). Fish with pre-B ALL (n=5, B1-5) showed GFPlo cells in each tissue, with GFPhi cells (i.e., T cells) present only in thymus. Two fish (B1, B2) had numerous thymic GFPlo cells, demonstrating extensive invasion. hMYC T-ALL controls (n=3, T1-3) exhibited near-exclusively GFPhi populations in every tissue. We also analyzed one mixed-ALL (M1). Consistent with two co-existing ALLs, the thymus, spleen, and muscle and viscera all contained large proportions of both GFPlo and GFPhi cells, with GFPlo cells outnumbering T-ALL cells in marrow and peripheral blood. To conclusively test whether GFPlo and GFPhi cells always equate to B-vs. T-lineage, we analyzed igic1s1 and rag1 in FACS-purified GFPlo and GFPhi cells from 5 of these fish (B1, B2, M1, T1, T2). As previously [Fig. 3A(e)], dim cells from every tissue expressed only igic1s1, and only bright cells were rag1+, including the GFPlo and GFPhi ALLs of M1. Thus, GFP reliably reflects pre-B vs. T-ALL in any niche, establishing hMYC;GFP zebrafish as a new and novel model to study both ALL types in one genetic context, or even one animal.
MYC-induced zebrafish pre-B and T-ALL have distinct expression signatures
To test whether D. rerio and human pre-B ALL share similar gene expression, we next defined GEPs in a new cohort of animals, quantifying 96 transcripts that distinguish B/T/NK cells, lymphoblasts, and precursor populations (genelist in Table S1) (29, 30). We FACS-purified 8 pre-B ALL, 4 T-ALL, and 2 ALL from a mixed-ALL fish (Fig. 6A-C; GFPlo or GFPhi populations in orange or blue, respectively), as well as control lymphocytes. For T cell controls, we isolated GFPhi thymocytes from three 10-fish cohorts of control hMYC and lck:GFP fish (hMYC thymus-GFPhi, WT thymus-GFPhi; Fig. 6D). As another T cell control, we pooled marrow, lymphoid-gated (31) GFPhi cells from these same 30 WT fish (WT marrow-GFPhi; Fig. 6E, blue). hMYC marrow lacked GFPhi cells (Fig. 6F), so these were not analyzed. For B cells, we purified GFPlo and GFP- marrow cells of the same 3 hMYC control cohorts (hMYC marrow-GFPlo, hMYC marrow-GFP-; Fig. 6F) and from marrow of 30 WT fish used for thymocyte preparations (WT marrow-GFPlo, WT marrow-GFP-; Fig. 6E).
Using barcoded gene-specific probes (Nanostring nCounter™), we quantified mRNA levels of 96 transcripts in all 29 samples. Unsupervised analysis clustered all B and T cell triplicate controls tightly (hMYC thymus-GFPhi, WT thymus-GFPhi, hMYC marrow-GFP-, hMYC marrow-GFPlo), proving high reproducibility of biologic replicates (Fig. 7A). Each of the 29 samples segregated unequivocally as B-or T-lineage, with every GFP-/GFPlo sample grouping as B-lineage (n=17), and GFPhi samples forming a T-lineage group (n=12). Every GEP clustered perfectly according to GFP expression, whether from control fish or fish with ALL, and irrespective of whether cells were obtained from thymus or marrow. Notably, GFP- and GFPlo cells from WT and hMYC animals all exhibited similar B-lineage profiles, with hMYC GFPlo B cell GEPs most similar to pre-B ALL (Fig. 7A).
Pre-B and T-ALL GEPs were distinct (Fig. 7B; Table S2 lists differentially-expressed genes), with the GFPhi ALL (green) and GFPlo ALL (yellow) of the mixed-ALL animal grouping as T-or pre-B ALL, respectively. In total, ∼60 homologous genes able to distinguish human (30) and zebrafish (29) B vs. T cells likewise categorized hMYC pre-B vs. T-ALL. Key classifier-genes (Fig. 7C) matched prior qRT-PCR results (Fig. 3A), with both ALL types showing comparable levels of rag2, hMYC, and shmt2, a known direct MYC target (Fig. S5A) (21), reinforcing that hMYC levels and activity are similar in this dual pre-B/T-ALL model. To further examine conservation of gene expression between both types of D. rerio and human ALL, we also tested whether human homologues of the differentially-expressed hMYC pre-B-vs. T-ALL genes could reliably classify ALL from the MILE1 study (Fig. 7D). This signature exhibited remarkable classification power, separating nearly all of these human ALL correctly. Homologues of several other hematopoietic stem/progenitor-or immature lymphocyte-specific genes showed no significant difference between hMYC ALL types (Fig. S5B).
Like ALL, control hMYC and WT thymocytes expressed more rag1 than B cell controls (Fig. 7E, top). From this result, we deduce higher rag1 in zebrafish T-ALL vs. pre-B ALL is unrelated to malignancy, but a normal feature of D. rerio lymphoblasts, although different from mammals. Notably, hMYC B cell controls showed higher rag2 than WT B cells, implying hMYC may expand the pre-B cell population (Fig. 7E, bottom). We also analyzed Ig heavy chain expression. Surprisingly, pre-B ALL expressed only ighz (Fig. 7F), an isotype unique to teleost fish that is functionally analogous to mammalian IgA (32). GFPlo hMYC B cells expressed ighm (Fig. 7G, left), so ighm+ pre-B ALL should be detectable in hMYC fish—but these were not found. Therefore, MYC may be oncogenic in only the ighz-lineage. Another B-lineage abnormality we noted is that hMYC GFPlo cells lacked ighd (Fig. 7G, right), so hMYC may suppress ighd transcription or repress this lineage, just as it seems to block the T cell lineage in hMYC marrow (Fig. 6F). Overall, hMYC dramatically perturbs zebrafish B cell development, inducing ALL in ighz+ B cells, and skewing both the ighm and ighd lineages.
Finally, we found substantially higher lmo2 in normal and malignant B vs. T cells (Fig. 7H), mirroring LMO2 findings in human B vs. T cells (33), which we independently confirmed in public human B and T cell data (Fig. S5C). This contradicts the assertion that MYC-driven zebrafish T-ALL emulates the human TAL1/LMO subtype (6). Instead, hMYC T-ALL actually had the lowest lmo2 levels of the 9 populations that were tested, including all T cell controls (Fig. 7H).
Discussion
Pre-B ALL is the most common pediatric cancer and kills more children than any other type (3), but no good zebrafish models exist for this important disease. Drug screens (17, 18), genetic studies (11, 12, 15) and stem cell discoveries (13, 14) were all made possible by zebrafish T-ALL models, and these and other approaches would likely be similarly fruitful with D. rerio pre-B ALL. Here, we describe the first robust zebrafish pre-B ALL model. Unexpectedly, young 3-6 month hMYC fish—used for years in several of the aforementioned T-ALL studies—also develop highly-penetrant pre-B ALL. Remarkably, this went unrecognized for over a decade. In terms of detecting pre-B ALL, dual-transgenic hMYC;GFP fish proved particularly valuable to our study, because their lck:GFP expression not only allowed pre-B ALL to be detected, but their differing GFP levels also distinguished pre-B and T-ALL in vivo. This dichotomy in lck expression extends to normal B and T cells as well, is corroborated by flow cytometry, and corresponds precisely to B-and T-lineage GEPs (Figs. 1E, 2-7, S1, S4). Consequently, even in fish with concomitant pre-B and T-ALL—which we believe are unique to this model—these cell lineages and ALL types, can be reliably separated for independent study.
Apart from the utility of lck:GFP in this system, hMYC pre-B ALL are powerful because they emulate this human disease in several ways: histology and organ involvement (Figs. 3-4, S3-4), lck levels (Fig. S2) and, most importantly, gene expression signature (Fig. 7). In fact, genes that differentiate zebrafish pre-B vs. T-ALL (Fig. 7B) classify human ALL also (Fig. 7D). The GEPs we obtained revealed many transcripts that distinguish pre-B vs. T-ALL in this model, but we also report a two-gene panel to categorize hMYC ALL that requires only igic1s1 and rag1, and this panel can be applied to any hMYC genetic background.
Interestingly, although RAG1 is expressed by mammalian B-and T-lymphoblasts, we find that only D. rerio T-lymphoblasts express rag1 highly, with levels >70-and >155-fold greater in normal T or T-ALL cells than in B or pre-B ALL cells, respectively (Figs. 3A(e), 5, 7C,E). A recent report of zebrafish B cell development despite low rag1 supports our observation (34). Apart from rag1, pre-B ALL expressed other classic B-lymphoblast genes like rag2, surrogate light chain components, pax5, cd79a/b, and others (Figs. 3-4, 7C, S4C, S5A). This was true for every dim ALL, including GFPlo mixed-ALL, so we conclude mixed-ALL are co-existing pre-B and T-ALL, and not biphenotypic ALL. Certainly, because hMYC can induce both ALL types, it remains possible that mixed-lineage biphenotypic ALL may arise in this system, but to date, our analyses of >40 dim ALL have failed to detect any that express T cell genes, suggesting that GFPlo ALL are always B-lineage, and GFPhi always represent T-ALL. In future work, studying both ALL types in one background—or one animal—presents new opportunities, like efforts to find cooperating genetic lesions unique to one type of ALL, lymphocyte lineage-specific drugs, or myriad other applications.
Our results also demonstrate multiple features of abnormal B and T lymphopoiesis in hMYC fish. Of interest, pre-B ALL GEPs closely matched the gene expression pattern of a recently-described ighz+ B cell population dubbed “fraction 2” (34), suggesting hMYC is oncogenically active in this cell population. Supporting this, every pre-B ALL we identified was ighz+ (Fig. 7F). Liu et al. postulated that the zebrafish ighm-lineage lacks a classic pre-B stage. If this is correct, it is logical that pre-B ALL only occurs in ighz+ cells, the lineage that has pre-B cells. We note that ighm+ cells do express lck (Fig. 7G), so ighm+ pre-B ALL should be GFP+ and detectable in this system. Yet ighm+ pre-B ALL were never detected, so we conclude they do not occur. In addition to our finding that ALL develops in only ighz+ cells, we also found that hMYC alters other B-lineages, with ighm and ighd both reduced in hMYC marrow (Fig 7G). Whether this is due to ighm and ighd transcriptional—or lineage—repression remains to be determined. Notably, T cells are also diminished in hMYC marrow (Fig. 6F), so hMYC alters both the B and T lymphocyte lineages, beyond inducing both pre-B and T-ALL.
Clearly, hMYC is leukemogenic to lymphocytes and perturbs zebrafish lymphocyte development. In future work, hMYC pre-B ALL can be used for classic zebrafish approaches like chemical and genetic screens, or in mechanistic studies probing hMYC biology in either ALL type. MYC is arguably the most clinically-relevant oncogene, important in many cancers besides ALL (35), but MYC’s contrasting actions in distinct neoplasias remain largely unexplored. We show hMYC fish provide a novel system to address this topic via studies of both human ALL types, using a single model.
Materials and Methods
Zebrafish Care and Fluorescent Microscopy Screening
Zebrafish housed in an aquatic colony at 28.5°C on a 14:10 hour light:dark circadian cycle and cared for according to protocols approved by the University of Oklahoma Health Sciences Center IACUC (12-066 and 15-046). For all procedures, fish were anesthetized with 0.02% tricaine methanesulfonate (MS-222). 3-6 month old hMYC;GFP fish of both genders were screened for abnormal GFP patterns, using a Nikon AZ100 fluorescent microscope. Low exposure (200 ms, 2.8X gain) and high exposure (1.5s, 3.4X gain) settings were used to obtain images with Nikon DS-Qi1MC camera. Images were processed with Nikon NIS Elements Version 4.13 software.
Fluorescence-Activated Cell Sorting (FACS) and Flow Cytometry Analysis
Cells from whole fish, excluding head regions, or specific organs were dissociated using a pestle and passed twice through 35 μm filters. GFP+ cells were collected from the lymphoid and precursor gates (31) using a BD-FACSJazz Instrument. GFP intensity defined characteristic peaks on either side of ∼102 on the GFP intensity axis of this instrument; this value was used to discriminate between GFPlo and GFPhi peaks. For percentage calculations, all GFP+ cells from 105 total events in the lymphoid/precursor gate were evaluated. GFP weighted mean fluorescence intensity (wMFI) was calculated using the following formula: [(MFI of GFPlo population x % of GFPlo cells) + (MFI of GFPhi population x % of GFPhi cells)]. Flow cytometric analyses performed using FlowJo software.
RNA extraction and quantitative real-time polymerase chain reactions
Total RNA was extracted using Trizol according to manufacturer instructions (Invitrogen). For qRT-PCR, 16 ng of total RNA was reverse transcribed using standard methodology. SYBR-Green qRT-PCR was performed using a CFX96 TouchTM Real-Time PCR Detection System (Biorad). Each cDNA sample was tested in triplicate. The 2-DeltaCt (Delta Ct = Ctexperimental gene-Cthousekeeping gene) method (36) was used to calculate the relative expression of each gene to housekeeping genes (β-actin and eef1a1l1).
RNA Microarrays
In vitro transcription, hybridization to Zebrafish Genome Arrays (#900487), and biotin-labeling performed according to manufacturer instructions (Affymetrix). Microarray (.CEL file) data generated using Affymetrix GeneChip Command Console Software, normalized against the entire dataset using the justRMA algorithm, and analyzed using R-Bioconductor (Version 3.4.1). Unsupervised and supervised hierarchical clustering were used to group specimens based on Euclidean distance and the Ward method. Differentially-expressed genes identified by shrinkage t-tests (37) with local false discovery rate (lfdr) used to correct p-values. A lfdr <0.05 was considered significant for differentially-expressed probe-sets. Public microarray data from normal human B and T cells at various maturational stages (27) and leukemia patients on the MILE stage 1 study (28) were used for gene expression analysis.
Histology and Immunohistochemistry (IHC)
H&E stains performed using standard methodology. IHC stains performed using 1:8000 dilutions of anti-GFP antibody (GeneTex, #GTX20290) at 37°C using a Ventana BenchMark XT instrument. Staining of GFP+ tissues performed using an Inview 3,3’-Diaminobenzidine (DAB) detection kit (Ventana Medical Systems).
Western Blot Analysis
FACS-purified GFPlo pre-B ALL and GFPhi T-ALL cells were homogenized in lysis buffer [(20mM Tris-HCl pH 8.0, 137mM NaCl, 2mM EDTA, 1% NP-40, and 10% Glycerol supplemented with Protease Inhibitor Cocktail (Sigma, #P8340)]. Total protein was resolved on a 4„10% gradient polyacrylamide gel and transferred to a nitrocellulose membrane (both BioRad) using transfer buffer (25mM Tris, 192mM glycine, and 20% methanol). Tris-buffered saline, 5% non-fat dry milk, and 0.05% Tween-20, used for blocking. Blots incubated overnight at 4°C with anti-GFP primary antibody (Santa Cruz, #sc-9996), followed by incubation with horseradish peroxidase-conjugated secondary antibody (BD). Immuno-reactive bands detected using ECL 2 western blot substrate (Pierce). Parallel incubation with anti-β-actin antibody (Abcam, #ab8227) used as a positive control.
RNAscope-ultrasensitive in situ hybridization (RNA ISH)
RNAscope (Advanced Cell Diagnostics-ACD) fluorescent-field ISH was used to detect hMYC, cd79b and lat mRNA in fish sections. Fixation, sectioning, and staining performed using the RNAscope Multiplex Fluorescent Detection Kit v2 (#323110), according to manufacturer instructions (https://acdbio.com/). RNAscope probes (ACD) used to specifically detect human MYC (#311761-C2), D. rerio cd79b (#511481) and lat (#507681). Probe labels (PerkinElmer) as follows: TSA-Plus-Cyanine-3 (#NEL744001KT) for hMYC (yellow fluorescence), TSA-Plus-Cyanine-5 (#NEL745001KT) for cd79b (red), and TSA-Fluorescein (#NEL701A001KT) for lat (green). Slides imaged and analyzed using Operetta High-Content Imaging System (PerkinElmer) and Harmony 4.1 software.
Nanostring nCounter Gene Expression Profiling
GEPs of FACS-purified GFPlo and GFPhi cell populations were quantified using a 96-gene Custom CodeSet according to manufacturer instructions (Nanostring nCounter Technologies). Genes quantified using an nCounter Digital Analyzer and analysed using nSolver v3.0 software. Background thresholds defined by counts from a no-RNA blank that were subtracted from each sample. Raw counts were normalized to spiked-in positive control probes and housekeeping genes (β-actin, eef1a1l1 and gapdh), as suggested by the manufacturer. nSolver t-tests used to compare groups and identify differentially-expressed genes (FDR ≤0.05).
Statistical analysis
GraphPad Prism 7 software used to calculate Spearman correlations and non-parametric Mann-Whitney t-tests for genes tested by qRT-PCR. Two-tailed 95% confidence intervals were used to determine significance, and significant differences are reported as p-values with p* <0.05, ** <0.01, *** <0.001 and **** <0.0001.
Author contributions
C.B. and J.K.F. conceived and designed the research study. C.B., C.F., J.L.R., and J.K.F. analyzed the data. C.B., G.P., M.M., S.T.A., and J.B.G. performed experiments. T.S. and R.R.M. performed histologic analyses and imaging. G.t.K., J.L.R., and J.K.F. contributed essential reagents, tools and/or funding. S.B. and L.B. assisted with data analyses. C.B. and J.K.F wrote the manuscript. All authors revised and approved the final manuscript.
Conflict of interest disclosure statement
Authors declare no conflict of interests.
Acknowledgments
We thank Megan-Malone Perez at OUHSC for zebrafish care, Sheryl Tripp at ARUP Laboratories for tissue sectioning and IHC, and Drs. Stephan Ladisch, David Jones, and Linda Thompson for critically reading the manuscript, and Dr. Nikolaus S. Trede for his support at the project’s outset. An Institutional Development Award (IDeA) from the National Institute of General Medical Sciences (P20 GM103639) supports the OUHSC Stephenson Cancer Center’s Histology and Immunohistochemistry Core, which performed services for this project. Financial support: J.K.F. received support from Hyundai Hope On Wheels, the Oklahoma Center for the Advancement of Science and Technology (HRP-067), and an INBRE pilot project award from the National Institute of General Medical Sciences (P20 GM103447), and holds the E.L. & Thelma Gaylord Endowed Chair of the Children’s Hospital Foundation.