JAK2 is dispensable for maintenance of JAK2 mutant B-cell acute lymphoblastic leukemias

Kim et al. show that while expression of mutant Jak2 is necessary for B-cell acute lymphoblastic leukemia induction, neither its continued expression nor enzymatic activity is required to maintain leukemia survival and rapid proliferation.

Relapsed B-cell acute lymphoblastic leukemia (B-ALL) is one of the leading causes of cancer-related mortality in children and young adults (Hunger and Mullighan 2015). Between 15% and 30% of all childhood B-ALLs, termed "Ph-like" B-ALL, exhibit a gene expression profile similar to that of BCR-ABL1-expressing B-ALL and are associated with poor prognosis (Den Boer et al. 2009;Mullighan et al. 2009b;Roberts et al. 2014). Little is currently known about the alternative treatment options in the up-front setting or recurrent disease for this cohort that is at high-risk of relapse, highlighting an urgent need for novel therapeutic strategies in this subset of B-ALL (Cario et al. 2010;Ensor et al. 2011).
Recent genomic studies revealed that Ph-like B-ALLs comprise a spectrum of somatic variations in genes encoding protein kinases and cytokine receptor signaling molecules (Roberts et al. , 2014. The majority of cases in Ph-like B-ALL harbors CRLF2 gene rearrangements (either IGH-CRLF2 or P2RY8-CRLF2) leading to cell surface overexpression of CRLF2, a component of the heterodimeric cytokine receptor complex for thymic stromal lymphopoietin (TSLP) (Mullighan et al. 2009a;Russell et al. 2009). Approximately 50% of CRLF2-rearranged B-ALLs have concomitant activating point mutations at highly conserved regions of JAK2 (exon 16), the most common of which is the B-ALL-exclusive activating JAK2 R683G alteration (Mullighan et al. 2009a;Harvey et al. 2010;Hertzberg et al. 2010).
Somatic JAK2 point mutations have been shown previously to be bona fide cancer-initiating lesions in myeloproliferative neoplasms (MPNs) (Baxter et al. 2005;Levine et al. 2005;Lacout et al. 2006;Wernig et al. 2006), where ∼95% of polycythemia vera (PV) and 50% of essential thrombocythemia (ET)/primary myelofibrosis (PMF) cases harbor the recurrent activating JAK2 V617F alteration (Baxter et al. 2005;James et al. 2005;Kralovics et al. 2005;Levine et al. 2005). This discovery provided a rationale for targeting deregulated JAK2 signaling as a treatment strategy in MPNs, prompting the rapid development and clinical application of type I (ATP-competitive) JAK2 kinase inhibitors (JAK2is), including Food and Drug Administration (FDA)-approved ruxolitinib (Verstovsek et al. 2012). Despite the lack of molecular response observed in the majority of ruxolitinib-treated MPN patients (Harrison et al. 2012;Verstovsek et al. 2012), genetic studies have demonstrated that MPN cells are indeed reliant on aberrant JAK/STAT signaling, underscoring its relevance as a therapeutic target in this disease (Bhagwat et al. 2014).
Mutant JAK2-expressing B-ALLs also exhibit deregulated JAK/STAT signaling , suggesting that inhibition of this hyperactive signaling network may be therapeutically beneficial. However, B-ALLs exhibit limited inherent sensitivity to structurally distinct type I JAK2is (including ruxolitinib) (Maude et al. 2012;Tasian et al. 2012), which induce paradoxical JAK2 Y1007/1008 activation loop hyperphosphorylation despite potent suppression of key JAK2 substrates, including STAT5 (Weigert et al. 2012;Wu et al. 2015). The putative maintenance of JAK2 signaling following type I JAK2i treatment of B-ALLs led to the development of alternative strategies to antagonize JAK2 kinase activity (Weigert et al. 2012;Wu et al. 2015). Accordingly, the type II (non-ATP-competitive) JAK2i CHZ868 that potently abrogates pJAK2 Y1007/1008 and downstream pSTAT5 Y694 was shown recently to exhibit efficacy superior to that of type I JAK2is in inducing apoptosis of CRLF2-rearranged/JAK2 mutant B-ALLs (Wu et al. 2015).
Here, we developed genetically engineered mouse models (GEMMs) of CRLF2/JAK2 mutant B-ALLs and used a pharmacogenomics approach to delineate the functional role of JAK2 in these leukemias and human CRLF2-rearranged/JAK2 I682F -expressing MHH-CALL4 B-ALLs. We demonstrate an essential role for mutant Jak2 in cooperating with overexpressed surface Crlf2 to initiate B-ALL development. However, pharmacological JAK2 inhibition using type I JAK2i ruxolitinib or genetic depletion or deletion of JAK2 resulted in only a marginal decrease in cell proliferation with little or no induction of cell death. Treatment of these leukemias with CHZ868 resulted in apoptosis (Wu et al. 2015); however, we demonstrate that this compound remained potent even in JAK2-depleted CRLF2/JAK2 mutant B-ALLs, indicating a putative off-target effector mechanism. Consequent interrogation for mechanisms underlying the maintenance of proliferation of CRLF2/mutant JAK2-driven B-ALLs following sustained genetic or pharmacological targeting of JAK2 revealed enhanced c-Myc protein expression and a compensatory c-Myc-driven gene expression signature that may have mediated prolonged growth of these cells. Target depletion of c-Myc in JAK2 mutant B-ALLs using the BET inhibitor (JQ1) or RNAi combined with direct inhibition of JAK2 using ruxolitinib potently abrogated c-Myc expression and synergistically killed CRLF2/JAK2 mutant B-ALLs. Cotreatment of mice bearing CRLF2/JAK2 mutant B-ALLs with JQ1 and ruxolitinib further prolonged overall survival of mice compared with single-agent treatment.

Pharmacological inhibition or genetic depletion of JAK2 in MHH-CALL4 B-ALL cells induces an anti-proliferative response and not apoptosis
We and others demonstrated previously that type I JAK2is can kill JAK2 V617F -driven MPNs (Supplemental Fig. S1A, B; Geron et al. 2008;Wernig et al. 2008), TEL-JAK2-driven T-cell ALLs (T-ALLs) (Waibel et al. 2013), JAK2-amplified classical Hodgkin lymphomas (cHLs), and primary mediastinal large B-cell lymphomas (Hao et al. 2014). However, these agents had limited activity against mutant JAK2expressing B-ALL cells, putatively due to paradoxical JAK2 Y1007/1008 hyperphosphorylation mediated by these agents in these cells (Weigert et al. 2012;Wu et al. 2015). We noted that while ruxolitinib treatment of JAK2 I682F -expressing MHH-CALL4 B-ALL cells did cause JAK2 Y1007/1008 hyperphosphorylation (Supplemental Fig. S1B), this agent had little or no effect on cell survival and suppressed cell proliferation only after 6-8 d of continuous culture with drugs ( Fig. 1A; Supplemental Fig. S1A). Interestingly, key downstream pathways of mutant JAK2 that regulate cell survival and proliferation such as STATs (STAT1 and STAT5), S6, and MEK/ERK were potently suppressed in ruxolitinib-treated MHH-CALL4 cells to levels equivalent to ruxolitinib-treated JAK2 V617Fexpressing human megakaryoblastic SET-2 cells (Supplemental Fig. S1B). Moreover, the JAK/STAT gene expression signature remained significantly down-regulated following ruxolitinib treatment of MHH-CALL4 cells, indicating the absence of compensatory maintenance of this pathway following JAK2 inhibition with a type I JAK2i (Supplemental Fig. S1C).
These results raised the possibility that the modest effects of type I JAK2is in JAK2 mutant B-ALL may not be due to paradoxical JAK2 Y1007/1008 hyperphosphorylation but rather may be due to the cells not being primarily reliant on mutant JAK2 for sustained survival and/or proliferation. To test this hypothesis, we transduced MHH-CALL4 cells with retroviral vectors expressing shRNAs targeting JAK2 (shJAK2.209 and shJAK2.2826) or a nontargeting shRNA (shSCR). JAK2 knockdown in MHH-CALL4 cells resulted in decreased phosphorylation of STAT1 Y701 and STAT5 Y694 (Fig. 1B) and significant negative enrichment of the JAK/STAT gene expression signature (Fig. 1C), mirroring the biochemical and molecular effects of ruxolitinib. Consistent with the effects of ruxolitinib on B-ALL cell proliferation, JAK2 knockdown did not induce apoptosis (Fig. 1D,I), and competitive growth assays demonstrated a loss of representation of JAK2depleted MHH-CALL4 cells evident after >8 d (Fig. 1E). In agreement with these findings, CRISPR-Cas9-mediated JAK2 deletion with two independent single guide RNAs (sgRNAs) resulted in little or no induction of cell death (Fig. 1F,G) but led to a loss of representation in a competitive proliferation assay (Fig. 1H). Additional cell  , 8, 9, 12, 14, 16, and 19 after transduction). (E) The percentage of shRNA + cells (GFP + ) was assessed by flow cytometry, and data were normalized to day 5. (F) Immunoblotting against the indicated target was performed against MHH-CALL4-Cas9 cells transduced with lentiviral vector (FgH1tUTG-GFP) expressing single guide RNAs (sgRNAs) targeting JAK2 (shJAK2.9901 or shJAK2.9903) at day 6 after sgRNA induction. β-Actin served as loading control. (G) Transduced populations from F were assessed for percentage Annexin V/DAPI positivity by flow cytometry at the indicated time points. (H) Transduced and nontransduced populations from F were passaged in vitro for 16 d. The percentage of sgRNA + cells (GFP + ) was assessed by flow cytometry (individual bars represent days 3, 5, 7, 9, 12, 14, and 16 after sgRNA induction). (I ) MHH-CALL4 cells were transduced with pLMS-shJAK2.209 or pLMS-shSCR. At day 14 after transduction, cell cycle analysis was performed by flow cytometric analysis of BrdU incorporation versus DNA content (7-AAD). Error bars in D, E, G, and I represent SEM (n = 2); error bars in H represent SEM (n = 3). ( * * ) P < 0.01; (ns) nonsignificant (P > 0.05).
cycle analysis of JAK2-depleted MHH-CALL4 cells showed a significant reduction in cells in S phase with a concomitant increase in G 0 /G 1 populations ( Fig. 1I; Supplemental Fig. S1D).
A recent report demonstrated that the type II JAK2i CHZ868, which stabilizes JAK2 in an inactive conformation, kills JAK2 mutant B-ALL cells (Wu et al. 2015). Consistent with that report, we observed potent apoptotic effects of CHZ868 without JAK2 Y1007/1008 hyperphosphorylation following culture in MHH-CALL4 cells (Supplemental Fig. S1E,F). However, CHZ868 was equipotent in parental MHH-CALL4 cells and those in which JAK2 had been effectively depleted and JAK2 downstream signaling was abrogated (Supplemental Fig. S1F), while ruxolitinib was effective only in JAK2-proficient cells, indicating that the apoptotic effects of CHZ868 were not reliant only on inhibition of JAK2 (Supplemental Fig.  S1F). Interestingly, despite their divergent phenotypic response, 3 ′ RNA sequencing (3 ′ RNA-seq) revealed that ruxolitinib did elicit a transcriptional response similar to that of CHZ868 in MHH-CALL4 cells (Supplemental Fig. S1G,H, dimension 1). However, principal component analysis (PCA) did unravel differences in gene expression mediated by ruxolitinib and CHZ868, which may highlight transcriptional changes induced by the JAK2-independent effects of CHZ868 (Supplemental Fig. S1H, dimension 2). Indeed, hierarchical clustering of these genes demonstrated that the ruxolitinib response more closely resembled the transcriptional changes observed following JAK2 knockdown than CHZ868 (Supplemental Fig. S1I). Gene ontology enrichment analysis of the genes altered by CHZ868 and not ruxolitinib or JAK2 depletion revealed enrichment of gene sets related to DNA damage response and DNA replication (Supplemental Fig. S1J). Collectively, these results suggest that the apoptotic effects observed following CHZ868 treatment of MHH-CALL4 B-ALLs are likely due to its JAK2-independent activities, while on-target pharmacological JAK2 inhibition or genetic depletion of JAK2 results in reduced cell proliferation mediated by a delay in transition from G 0 /G 1 to S phase of the cell cycle (Supplemental Fig. S1D).

Crlf2 and mutant Jak2 cooperate to induce murine B-ALL development in vivo
The frequent co-occurrence of CRLF2-rearrangements and activating JAK2 mutants in B-ALL suggest that these events functionally cooperate in disease pathogenesis (Mullighan et al. 2009a;Harvey et al. 2010;Hertzberg et al. 2010). To more accurately model CRLF2-rearranged/ JAK2 mutant B-ALL, we initially generated transgenic mice to mimic the recurrent IGH-CRLF2 and P2RY8-CRLF2 translocations seen in human B-ALL (Mullighan et al. 2009a;Russell et al. 2009). A transgene consisting of a wild-type Crlf2 cDNA sequence inserted downstream from the Eµ/SRα promoter/enhancer elements was used to generate transgenic mice with high-level transgene expression (Eµ-Crlf2) exclusively in B-lineage hematopoietic cells ( Fig. 2A; Bodrug et al. 1994). Flow cytometric analy-sis of surface Crlf2 expression confirmed B-cell (B220 + )specific high-level transgene expression in the peripheral blood, spleens, and bone marrow of Eµ-Crlf2 transgenic mice (Supplemental Fig. S2A). Eµ-Crlf2-mediated overexpression of Crlf2 alone did not constitutively activate downstream pStat5 Y694 (Supplemental Fig. S2B), and these mice did not develop B-ALL by 18 mo of age (data not shown), demonstrating that Crlf2 overexpression alone was insufficient to induce B-cell leukemia.
All Dox-treated (Jak2-off) recipient mice harboring Jak2 knockdown-persistent cells exhibited a delay in leukemia progression and conferred a significant survival benefit over the untreated (Jak2-on) cohort (Fig. 4B). Flow cytometric and Western blot analysis confirmed sustained Jak2 shRNA expression (determined by detection of dsRed) and concomitant depletion of Jak2 in splenic cells harvested from moribund mice in the Dox-on (Jak2-off) group (Fig. 4C,D). In contrast, Jak2 knockdown persistent cells harvested from moribund mice in the Dox-off group re-expressed Jak2 and showed concomitant loss of dsRed expression (Fig. 4C,D), and mice in this cohort succumbed to disease more rapidly than mice in the Dox-treated (Jak2-off) group (Fig. 4B). These data indicate that Eµ-Crlf2/Jak2 mutant B-ALLs with sustained knockdown of Jak2 retain their immortality and continue to proliferate, albeit at a reduced rate. Thus, Jak2 expression is not necessary for these cells to persist in vivo.

JAK2 is dispensable for maintenance of B-ALLs
Genome-wide transcriptome analysis of Jak2 knockdown persistent Eμ-Crlf2/Jak2 R683G -driven B-ALLs We hypothesized that Eµ-Crlf2/Jak2 mutant B-ALLs that maintained proliferative capacity following sustained (chronic) Jak2 depletion developed compensatory cell growth mechanisms that would be apparent when comparing gene expression signatures in these cells with parental cells and cells subjected to only short-term (acute) Jak2 depletion. RNA-seq was performed on Eµ-Crlf2/ Jak2 R683G B-ALLs that were subjected to 2 d (acute Jak2 knockdown) or 21 d (chronic Jak2 knockdown) of RNAimediated Jak2 knockdown in vivo (Fig. 5A). Consistent with our hypothesis, global transcriptome analysis using two distinct methodologies (PCA and unsupervised hierarchical clustering of all differentially expressed genes) confirmed that naïve Eµ-Crlf2/Jak2 R683G B-ALL cells (control) and those with acute or chronic knockdown of Jak2 all displayed distinct transcriptional profiles, and their respective replicates clustered identically (Fig. 5B,C). To identify putative "adaptive pathways" apparent in Eµ-Crlf2/Jak2 R683G leukemia cells with chronic relative to acute Jak2 depletion, gene set enrichment analysis (GSEA) was performed against all publicly available oncogenic signature data sets (C6, Broad Institute Molecular Signatures Database [MSigDB]) (Subramanian et al. 2005). Strikingly, a significant positive enrichment for the MYC gene signature was apparent in Eµ-Crlf2/ Jak2 R683G leukemia cells with chronic Jak2 depletion across multiple data sets (FDR < 0.01) ( Fig. 5D; Supplemental Fig. S4A). In addition, GSEA demonstrated up-regulation of multiple gene sets containing c-Myc-binding sites (Supplemental Fig. S4B). There was no up-regulation of gene sets containing STAT5A-binding sites (Supplemental Fig. S4B), indicating that Eµ-Crlf2/Jak2 R683G B-ALLs that persisted despite sustained depletion of Jak2 did not do so by reactivating STAT5A target genes. Analysis of enriched motifs in promoters of genes induced upon chronic relative to acute Jak2 depletion revealed c-Myc as the top-ranked transcription factor (P < 0.001) (Supplemental Fig. S4C). Finally, transcriptome-wide analysis using a larger c-Myc target gene set (HALL-MARK_MYC_TARGETS_V1) revealed that the expression of a large number of these genes displayed enhanced expression in Eµ-Crlf2/Jak2 R683G B-ALL cells with sustained (chronic) Jak2 depletion (Fig. 5E).
Myc transcript levels in chronic Jak2 knockdown Eµ-Crlf2/Jak2 R683G cells were not significantly elevated relative to acute Jak2 knockdown cells (Fig. 5F). However, consistent with the apparent global up-regulation of Myc target genes in chronic Jak2 knockdown Eµ-Crlf2/ Jak2 R683G cells, c-Myc protein levels were prominently up-regulated in Eµ-Crlf2/Jak2 R683G leukemia cells following sustained Jak2 depletion (shJak2.3323, C#8 M7 and M8) (Fig. 5G). This indicates that enhanced signaling through c-Myc in Eµ-Crlf2/Jak2 R683G leukemia cells following chronic Jak2 depletion is mediated by enhanced c-Myc protein levels in the absence of any increase in mRNA levels. Myc expression remained unchanged when inhibition of Jak2 was relieved in tertiary recipients of chronic Jak2 knockdown Eµ-Crlf2/Jak2 R683G cells (Supplemental Fig. S4D) despite mice in this cohort succumbing to disease more rapidly than mice in which Jak2 knockdown was maintained (Fig. 4B).
We next assessed whether JAK2 mutant human B-ALLs exhibited similar compensatory proliferative mechanisms following prolonged JAK2 depletion. MHH-CALL4 cells were transduced with pLMS-GFP vectors expressing control (shSCR) or JAK2 targeting (shJAK2.209 or shJAK2.2826) shRNAs, and cells were harvested at day 4 (acute JAK2 knockdown) or day 10 (chronic JAK2 knockdown) for immunoblotting analysis. MHH-CALL4 cells with acute depletion of JAK2 using two independent JAK2 shRNAs had reduced c-Myc expression (Fig. 5H). However, c-Myc expression was prominently up-regulated following chronic JAK2 knockdown (Fig. 5H), similar to the result seen in Eµ-Crlf2/Jak2 R683G B-ALLs, demonstrating similar compensatory proliferative mechanisms across the two models. Taken together, these data demonstrate up-regulation of c-Myc and global amplification of its target genes following sustained Jak2 knockdown in B-ALLs driven by oncogenic Jak2.

Combined targeting of JAK2 and c-Myc is effective against CRLF2/JAK2 mutant B-ALLs
Small molecule BET bromodomain inhibitors (BETis) have been shown previously to suppress c-Myc mRNA and expression of its target genes in a range of tumors, including JAK2 mutant B-ALLs (Dawson et al. 2011;Delmore et al. 2011;Zuber et al. 2011b;Ott et al. 2012). MHH-CALL4 cells expressing control shRNA (shSCR) or two JAK2 targeting shRNAs (shJAK2.209, shJAK2.2826) were harvested and cultured with BETis after 10 d of culture when knockdown of JAK2 was evident (Supplemental Fig. S5A). Interestingly, while MYC levels were equivalently suppressed in all three cell lines (Fig. 6A), only MHH-CALL4-pLMS-shJAK2.209 and MHH-CALL4-pLMS-shJAK2.2826 cells with chronic (10-d) depletion of JAK2 demonstrated sensitivity to BETi-induced apoptosis (Fig. 6B). These results demonstrate that MHH-CALL4 cells with sustained depletion of JAK2 demonstrate a synthetic-lethal response to BETi-mediated down-regulation of MYC. The potent effects of dual targeting of JAK2 and c-Myc in MHH-CALL4 cells were further demonstrated using pharmacological agents ruxolitinib and JQ1 (Fig. 6C). In addition to the enhanced apoptotic response to the combination of ruxolitinib and JQ1, these agents provided superior down-regulation of c-Myc when combined compared with either single agent (Fig. 6D). Congruent with these findings, chronic c-Myc-depleted MHH-CALL4-pLMS-shMYC.1891 cells demonstrated enhanced sensitivity to ruxolitinib relative to MHH-CALL4 cells expressing the control shRNA (Fig. 6E), and the combination of ruxolitinib and c-Myc knockdown induced superior down-regulation of c-Myc compared with either knockdown or ruxolitinib alone ( Fig. 6F; Supplemental Fig. S5B). Intriguingly, c-Myc knockdown in MHH-CALL4 cells resulted in reduced phosphorylation and expression of STAT5 (Fig. 6F) reminiscent of JQ1 treatment, which down-regulates surface IL-7R expression and leads to reduced STAT5 Y694 phosphorylation in this context ( Fig. 6D; Ott et al. 2012). However, flow cytometric analysis revealed no significant differences in surface expression of either IL-7R or CRLF2 in MHH-CALL4-pLMS-shMYC.1891 cells relative to MHH-CALL4-pLMS-shRenilla.713 cells (Supplemental Fig. S5C).
To assess the in vivo potential of concurrent JAK2 and c-Myc inhibition in vivo, mice were transplanted with Eµ-Crlf2/Jak2 R683G B-ALL cells and treated with 50 mg/ kg JQ1 once per day and 90 mg/kg ruxolitinib twice per day alone or in combination. After 3 d of drug treatment, mice receiving the combination had significantly lower peripheral WBC count, marked reductions in splenomegaly, and reduced splenic tumor burden (assessed by percentage GFP positivity) compared with mice treated with vehicle or either single agent (Fig. 6G,H). The biochemical effects seen in MHH-CALL4 cells (Fig. 6D) following single-agent and combination treatment with JQ1 and ruxolitinib were also observed in Eµ-Crlf2/ Jak2 R683G B-ALL cells following in vivo treatment with these agents (Fig. 6I), highlighted by the almost complete loss of c-Myc following combination treatment. Additional cohorts of mice transplanted with Eµ-Crlf2/Jak2 R683G B-ALL cells were treated for 3 wk with JQ1 and/or ruxolitinib. Both single-agent ruxolitinib and JQ1 increased overall survival compared with vehicle treatment (Fig.  6J), while the combination therapy, which was well-tolerated (Supplemental Fig. S5D-F), further prolonged overall survival, with an improvement in median survival from 18 d in vehicle-treated mice to 28 d in mice following combination treatment (Fig. 6J).

Discussion
The discovery of mutant constitutively active JAK2 in high-risk B-ALL highlighted JAK2 as a promising novel therapeutic target in this disease. However, preclinical studies showed that type I JAK2is induce persistent JAK2 Y1007/1008 hyperphosphorylation and exhibit limited efficacy against JAK2 mutant B-ALLs (Weigert et al. 2012;Wu et al. 2015). A phase II clinical trial is currently under way testing ruxolitinib in CRLF2/JAK2 mutant Phlike B-ALL, further highlighting the critical need to elucidate the relevance of JAK2 as a therapeutic target in this disease (ClinicalTrials.gov identifier: NCT02723994).
Here, we developed novel GEMMs of human B-ALL through transgenic overexpression of Crlf2 and concomitant expression of mutant Jak2 to determine the role of mutant JAK2 in leukemia initiation and maintenance of disease. We initially showed that Crlf2 and mutant Jak2 alone (Jak2 R683G and Jak2 P933R ) were insufficient for B-ALL induction using the fetal liver transplantation approach, congruent with previous findings using transgenic Eµ-JAK2 R683G mouse models (Lane et al. 2014). Moreover, we demonstrated that while mutant Jak2 can cooperate with Crlf2 to drive development of leukemia in mice, genetic depletion or pharmacological inhibition of Jak2 served merely to delay proliferation of the leukemias, resulting in a modest therapeutic response in vivo. CRLF2/JAK2 mutant B-ALL cells that remained viable following long-term depletion or pharmacological inhibition of JAK2 developed an adaptive response that featured enhanced expression of c-Myc and downstream Myc target genes. Importantly, this adaptive response revealed a synthetic lethality to both RNAi-and BETi-associated down-regulation of c-Myc expression, forming the rationale for combining BETis with JAK2i ruxolitinib, which proved to be a superior therapeutic strategy in vitro and in vivo compared with either single-agent treatment.
Our study challenges the notion that type I JAK2is such as ruxolitinib are relatively ineffective for the treatment of CRLF2/JAK2 mutant leukemia due to paradoxical JAK2 Y1007/1008 hyperphosphorylation (Weigert et al. 2012;Wu et al. 2015). While our new data and previously published reports (Weigert et al. 2012;Wu et al. 2015) clearly demonstrate that ruxolitinib does induce JAK2 hyperphosphorylation in JAK2 mutant B-ALLs, it is also clear that key downstream targets of JAK2 such as STAT1, STAT3, STAT5, S6, and ERK remain in an inactive state with suppression of the STAT5-dependent gene expression signature. While the type II JAK2i CHZ868 did effectively kill CRLF2/JAK2 mutant B-ALL cells without inducing JAK2 Y1007/1008 hyperphosphorylation, this agent remained effective even in cells in which the putative molecular target (JAK2) had been genetically depleted. This therefore raises questions regarding the offtarget effects of the CHZ868 type II inhibitor that require further comprehensive evaluation and more broadly about the importance of mutant JAK2 in maintaining cell proliferation or survival in JAK2 mutant B-ALLs.
Using both established human CRLF2/JAK2 mutant B-ALL cells and primary mouse Eµ-Crlf2/Jak2 R683G or Eµ-Crlf2/Jak2 P933R leukemias, we showed that depletion or inhibition of Jak2 did not affect the viability of these cells but resulted in fewer leukemic cells entering into the cell cycle, which was reflected by a modest therapeutic response in vivo. Our findings were congruent with various other studies demonstrating minimal efficacy of singleagent ruxolitinib in multiple independent JAK2 mutant ALL patient-derived xenograft (PDX) models and the lack of cytotoxicity observed in JAK2 mutant human cell lines performed in short-term in vitro assays (Maude et al. 2012;Tasian et al. 2012;Weigert et al. 2012;Steeghs et al. 2017). Eµ-Crlf2/Jak2 mutant B-ALL cells that had remained viable even weeks after knockdown of Jak2 in vivo retained leukemogenic potential and remained partially sensitive to Jak2 depletion, as shown by serial transplant experiments. However, when Jak2 expression was relieved in these cells, the leukemias were more aggressive, indicating that oncogenic signaling pathways downstream from mutant Jak2 remained intact in these cells.
How, then, do CRLF2/JAK2 mutant B-ALL cells that depend on mutant JAK2 for disease initiation remain alive and proliferating (albeit more slowly) following sustained pharmacological inhibition or genetic depletion of the driving oncoprotein JAK2? We have no biochemical or molecular evidence to suggest that JAK/STAT signaling had been restored in leukemias with sustained inhibition or depletion of JAK2. This is in contrast to studies using JAK2 V617F mutant SET-2 megakaryoblastic leukemia cells that acquire resistance following prolonged culture in ruxolitinib (Koppikar et al. 2012). In that system, ruxolitinibresistant SET-2 cells maintained JAK/STAT signaling through compensatory heterodimeric activation of JAK2 by other JAK family members such as JAK1 and TYK2, and RNAi-mediated depletion of JAK2 resulted in decreased viability of the resistant populations (Koppikar et al. 2012). Interestingly, it is apparent that SET-2 myeloproliferative disorder-derived megakaryoblastic leukemia cells expressing JAK2 V617F undergo rapid ruxolitinib-mediated apoptosis (Geron et al. 2008;Wernig et al. 2008) as opposed to the mild suppression in cell growth seen in ruxolitinib-treated CRLF2/JAK2 mutant B-ALL cells that we demonstrated here. The difference in biological response to ruxolitinib seen on the different JAK2 mutant cells occurs even though ruxolitinib equivalently suppressed phosphorylation of STAT1, STAT5, MEK/ ERK, and S6 and induced JAK2 Y1007/1008 hyperphosphorylation in all cells (Supplemental Fig. S1B). These data indicate that JAK2 Y1007/1008 hyperphosphorylation is not a reliable biomarker for response to ruxolitinib in preclinical models and that dephosphorylation of downstream JAK2 targets, including STATs, MEK/ERK, and S6, is not sufficient to potently kill all JAK2 mutant malignancies.
Our study provides novel molecular insights into how JAK2 mutant B-ALL cells adapt to depletion or inhibition of JAK2 to maintain proliferative and leukemic potential. We discovered positive enrichment of the Myc gene expression signature and up-regulation of c-Myc protein expression in CRLF2/JAK2 mutant B-ALL cells subjected to prolonged JAK2 depletion. At present, it is unclear how c-Myc expression is enhanced in CRLF2/ JAK2 mutant B-ALL cells with sustained depletion/inhibition of JAK2; this remains to be elucidated in future studies, but the response did not appear to be driven by transcriptional up-regulation of c-Myc expression. It is possible that post-translational modifications of c-Myc, including phosphorylation, acetylation, and/or ubiquitination (Li et al. 2001;Adhikary and Eilers 2005;Faiola et al. 2005), may have contributed to its increased expression in CRLF2/JAK2 mutant B-ALL cells with sustained depletion or inhibition of JAK2 through one or more of the various signal transduction pathways that are known to regulate c-Myc expression (Vervoorts et al. 2006). Interestingly, we observed further interplay between c-Myc and the JAK/STAT signaling pathway in which phosphorylation and expression of STAT5 were suppressed upon c-Myc knockdown in MHH-CALL4 cells. We hypothesized that this may be due to the modulation of surface CRLF2 and/or IL-7R expression, reminiscent of prior studies demonstrating JQ1-induced IL-7R down-regulation and concomitant suppression of pSTAT5 (Ott et al. 2012). However, our data suggest that this association is independent of either surface CRLF2 or IL-7R expression modulation, thus warranting further investigations to elucidate the precise mechanism underlying this intriguing interplay between c-Myc and the JAK/STAT pathway in JAK2 mutant B-ALLs.
Interestingly, CRLF2/JAK2 mutant B-ALL cells with sustained depletion/inhibition of JAK2 generated a "neodependency" on c-Myc signaling, shown through the enhanced sensitivity of these cells to both RNAi-mediated depletion of c-Myc and the BET bromodomain inhibitors iBET151 and JQ1. Consistent with a previous report using a xenograft model of CRLF2-rearranged B-ALL, JQ1 provided a survival advantage to mice transplanted with syngeneic CRLF2/JAK2 mutant B-ALLs (Ott et al. 2012); however, this effect was augmented through coadministration of JAK2i ruxolitinib, which together demonstrated potent repression of c-Myc and induction of apoptosis in vitro. These results provide evidence that this well-tolerated combination approach may be an effective therapeutic strategy to treat JAK2 mutant B-ALLs and thus provide impetus for further preclinical and clinical testing of this combinatorial regimen.

Compounds
Ruxolitinib and CHZ868 were provided by Novartis. (+)-JQ1 (JQ1) and iBET-151 were kindly provided by James Bradner (Dana Farber Cancer Institute, Boston, MA). For in vitro use, compounds were dissolved in dimethylsulfoxide (DMSO) (Sigma-Aldrich) at a final stock concentration of 10 mM (stored at −20°C) and diluted to the appropriate concentration from stock prior to use. For in vivo application, ruxolitinib was dissolved and administered orally (oral gavage) in 0.5% methylcellulose solution (Sigma-Aldrich) at 90 mg/kg twice daily for the indicated duration. JQ1 was reconstituted in one part DMSO to nine parts 10% (w/v) hydroxypropyl-β-cyclodextrin (HPBCD) (Sigma-Aldrich) solution and administered via intraperitoneal injection at 50 mg/kg once daily for the indicated duration.

In vivo mouse experimentation
All experimental mice used in this study were housed in the animal facility at the PMCC (Melbourne, Australia). All procedures were conducted according to the PMCC Animal Experimental Ethics Committee guidelines (animal ethics application approval number E533) and in compliance with the NHMRC (National Health and Medical Research Council) "Australian Code of Practice for the Care and Use of Animals for Scientific Purposes." For transplantation of Eµ-Crlf2/Jak2 mutant B-ALLs in vivo, cohorts of 6-to 8-wk-old male NSG or C57BL/6 CD45.1 + wild-type mice were inoculated via tail vein injection with 2.5 × 10 5 to 5.0 × 10 5 Eµ-Crlf2/Jak2 mutant cells (in PBS). For shRNA induction in vivo, mice received Dox-supplemented drinking water (2 mg/mL with 2% sucrose; Sigma-Aldrich) and 625 mg/kg food (Specialty Feeds) for the specified time points. Transplanted mice were monitored weekly for leukemia development by weekly analysis of circulating leukemia cells (by flow cytometry) and assessment of total WBCs (Sysmex hematology analyzer, Sysmex Corporation) from peripheral blood. In all in vivo experiments, moribund bound mice were determined by animal technicians that were blinded of the treatment groups. For a detailed description of the fetal liver transplantation model, please see the Supplemental Material.

Cell viability and proliferation assays
For cell viability assays, cells were resuspended in 150 µL of Annexin V-binding buffer (10.0 mM HEPES at pH 7.4, 140.0 mM NaCl, 5.0 mM CaCl 2 in dH 2 O) containing 1:100 APC-conjugated Annexin V (BD Pharmingen), 1.0 µg/mL propidium iodide (Sigma-Aldrich), and/or 1.0 µg/mL DAPI (Sigma-Aldrich) and analyzed by flow cytometry in a 96-well plate (BD FACSVerse). Total viable cell counts were calculated using viable cell concentration data (volumetric cell counting). For proliferation assays, MHH-CALL4 cells were stained with CellTrace Violet (CTV) (Thermo Fisher) as per the manufacturer's instructions, FACSsorted to a single population (BD FACS Aria III) of CTV + cells, and assessed for CTV expression every 48 h by flow cytometry (BD LSR II flow cytometer). For details on BrdU/7-AAD cell cycle analysis, see the Supplemental Material.

Retroviral transduction and competitive proliferation assays
Retroviral pLMS-GFP/eBFP2 or pREBIR-eBFP2 (see the Supplemental Material for details on plasmid and shRNA sequence design) RNAi constructs were cotransfected with an appropriate packaging vector (pCL-Eco or pCL-Ampho) into HEK293T cells using calcium phosphate as described previously (Newbold et al. 2013). Viral supernatants were centrifuged onto RetroNectin-coated (30 µg per well; Takara, Clontech) six-well plates followed by spinfection of leukemia cells. The transduction efficiency in MHH-CALL4 cells was assessed 72 h after infection by flow cytometry (GFP or eBFP2 expression), and competitive proliferation assay was performed as described previously (Zuber et al. 2011a). For in vivo shRNA-mediated knockdown studies, Eμ-Crlf2/Jak2 mutant cells were injected intravenously into NSG mice 8 h after retroviral transduction. Established tumors were harvested, and eBFP2-positive cells were isolated by flow cytometry and retransplanted into NSG recipients by intravenous injection to obtain a pure population of transduced cells for experimentation.
CRISPR-Cas9-mediated gene knockout sgRNA oligonucleotides (Sigma-Aldrich) targeting JAK2 were annealed and cloned into lentiviral expression vector FgH1tUTG (Addgene, 70183; a gift from M. Herold) for inducible sgRNA expression (see the Supplemental Material for sgRNA sequences). Cells were first transduced with the Cas9 expression vector FUCas9Cherry (Aubrey et al. 2015), selected for mCherry-positive cells, and subsequently transduced with a lentiviral sgRNA expression vector. Cells were treated with 1.0 µg/mL Dox for transient sgRNA expression.

Histology
Leukemia-infiltrated livers and spleens from mice were resected and fixed in 10% neutral-buffered formalin (NBF) for at least 24 h prior to embedding tissue blocks in paraffin. Tissue processing, embedding, paraffin sectioning of tissues, and staining (hematoxylin and eosin; H&E) were performed by the Advanced Histology Platform (PMCC). All images were captured using the BX61 microscope (Olympus) at the Advanced Histology Platform (PMCC).
3 ′ RNA-seq/RNA-seq library preparation and sequencing Total RNA for 3 ′ RNA-seq and RNA-seq was freshly extracted using the NucleoSpin RNA isolation kit as per the manufacturer's instructions (Macherey-Nagel). The quantity and integrity of the resulting total RNA were assessed on the Agilent 2100 Bioanalyzer or TapeStation  DNA PCR assays PCR for detection of genomic recombination across distal V H 558 or proximal V H 7183, V H Q52 regions of the IgH locus was performed using 100 ng of genomic DNA for each reaction using degenerate primers from the distal V H 558 (5 ′ -CGAGCT CTCCARCACAGCCTWCATGCARCTCARC-3 ′ ) or proximal V H 7183 (VH7183 5 ′ -CGGTACCAAGAASAMCCTGTWCCTG CAAATGASC-3 ′ ) and V H Q52 (VQ52 5 ′ -CGGTACCAGACTG ARCATCASCAAGGACAAYTCC-3 ′ ) regions to the J3 segment (5 ′ -GTCTAGATTCTCACAAGAGTCCGATAGACCCTGG-3 ′ ) as described (Schlissel et al. 1991). Briefly, samples underwent denaturation for 3 min at 94°C followed by 33 cycles of 30 sec at 94°C, 30 sec at 60°C, and 1 min at 72°C and a final 10-min extension at 72°C. PCR products were separated by agarose gel electrophoresis and visualized with ethidium bromide staining.

Statistical analysis
All analyses were performed in GraphPad Prism unless specified (GraphPad Software, Inc.), and, for all statistical tests, P < 0.05 was considered statistically significant. For analyses limited to two groups, the Student's t-test was used, and statistical significance was calculated using the Holm-Sidak method. For multiple comparisons, one-way analysis of variance (ANOVA) with Tukey's statistical hypothesis testing method was used. All Kaplan-Meier survival curves were compared using the log-rank (Mantle-Cox) test. All flow cytometry data were analyzed using FlowJo analysis software version 10.2 (Treestar).