Cancer genome copy number changes are opportunistic, preferentially altering chromosomal regions that provide the greatest selective advantage for the malignant clone. This principle is exemplified by a recurrent chromosome amplicon in PMBL and HL that does not focus on a single gene but rather on a several megabase region on chromosome band 9p24. Using a functional genomics screen, we discovered that three amplicon genes – JAK2, JMJD2C, and RANBP6 – are required for the proliferation and survival of lymphoma lines bearing this amplicon. These genes are not essential to human cells in general since lymphoma lines lacking this amplicon were not dependent upon these genes. It thus appears that amplification of this genomic region creates a simultaneous addiction to these three genes. In some lines, inactivation of any one of these genes was toxic. In others, the simultaneous inactivation of JAK2 and JMJD2C was required to efficiently kill the cells. Our results thus demonstrate that a cancer amplicon can harbor more than one “driver” gene, and suggest that functional genomics will be required to gain a full understanding of the multiple addictions created by amplicons. This understanding may in turn lead to the rational combination of therapeutic agents targeting these addictions.
Although JAK2 is amplified in both PMBL and HL, mutations such as those in myeloproliferative disorders have not been found in these lymphoma types (Melzner et al., 2006
; Wu et al., 2009
). Rather, our data suggest that wild type JAK2 is activated by autocrine IL-13 signaling in these lymphomas and that the 9p24 amplicon increases signal strength through this pathway. STAT6 activation was blocked in all PMBL and HL lines treated with an anti-IL-13 antibody, and IL13Rα knockdown had a similar effect. IL-13 signaling in PMBL and HL cells up-regulated expression of IL13Rα, thereby creating a positive feed-forward loop. Perhaps as a result, expression of IL13RA1
mRNA is a hallmark of PMBL and HL that distinguishes them from other lymphoma types (Rosenwald et al., 2003
; Savage et al., 2003
). Moreover, IL4R
is a direct target of JAK2 histone phosphorylation in PMBL, leading to increased expression of IL4Rα, a subunit of the IL-13 receptor that significantly increases its affinity for IL-13.
Remarkably, one sixth of the genes that are characteristically expressed in PMBL tumors relative to GCB DLBCL tumors were activated by JAK2 signaling in a PMBL line. These JAK2-regulated genes were more highly expressed in PMBL tumors even in the absence of the 9p24 amplicon, suggesting that autocrine IL-13 signaling and JAK2 activation takes place in the absence of JAK2 amplification. However, the 9p24 amplicon further increased expression of these JAK2-regulated genes suggesting that one or more genes within the 9p24 amplicon augment the signaling output of the JAK2 pathway. Thus, JAK2 signaling has a defining influence on the biology of this lymphoma subtype that is aided and abetted by the 9p24 amplicon.
The cooperation between JAK2 and the histone demethylase JMJD2C suggests that JAK2 mediates its oncogenic effect in PMBL and HL by modulating the epigenome. Classically, JAK signaling mediates its biological effects by phosphorylating STAT transcription factors that then transactivate target genes bearing STAT binding motifs (Ghoreschi et al., 2009
). This signaling pathway undoubtedly plays a role in modulating the gene expression profile of PMBL and HL cells. However, of the genes that were most downmodulated in expression upon JAK2 inhibition in PMBL (n=1701) and HL (n=1027), only 2.5% contain canonical STAT6 binding sites in their regulatory regions (data not shown). Thus, much of the biology of PMBL and HL cells that is controlled by JAK2 is likely to come from other regulatory mechanisms. Studies in Drosophila
(Shi et al., 2006
; Shi et al., 2008
) and human leukemia (Dawson et al., 2009
) have highlighted the ability of JAK signaling to globally decrease heterochromatin formation. In our study, JAK2 cooperated with the histone demethylase JMJD2C in several assays, suggesting that epigenetic modulation by JAK2 is a key aspect of its oncogenic action in lymphomas bearing the 9p24 amplicon. Specifically, inhibition of JAK2 and JMJD2C cooperatively killed PMBL and HL lines, increased genome-wide histone H3K9me3 levels, and promoted heterochromatin formation. Moreover, inhibition of JAK2 and JMJD2C cooperated to repress MYC expression, which was associated with remodeling of the MYC
locus by two hallmarks of heterochromatin, H3K9me3 and HP1α recruitment.
Heterochromatin has been conceptually subdivided into stable “constitutive” heterochromatin and dynamic “facultative” heterochromatin (reviewed in (Trojer and Reinberg, 2007
)). The local epigenetic modification that we observed at the MYC
locus is most reminiscent of the facultative heterochromatin state, such as is mediated by the Rb protein, which represses the S-phase gene cyclin E
during G1 phase by recruiting a histone H3K9 methyltransferase, leading to HP1 recruitment (Nielsen et al., 2001
). On the other hand, JAK2 and JMJD2C inhibition was associated with a microscopically discernable increase in HP1α-associated nuclear speckles. Previous work has shown that the chromatin in these nuclear domains recruits HP1α by possessing H3K9me3 marks and lacking H3Y41 phosphorylation (Dawson et al., 2009
). These nuclear domains may represent the formation of stable foci of constitutive heterochromatin or alternatively may represent the reversible recruitment of genes such as MYC
to nuclear regions where gene silencing occurs.
Our working model of the epigenetic cooperation between JAK2 and JMJD2C is shown in . Both regulators control recruitment of the heterochromatin protein HP1α to histone tails, but by different mechanisms. HP1α uses its chromo domain to bind histone H3K9me3 (Bannister et al., 2001
; Lachner et al., 2001
), and demethylation of this residue by JMJD2C removes this HP1α binding site (Cloos et al., 2006
; Loh et al., 2007
; Whetstine et al., 2006
; Wissmann et al., 2007
). HP1α uses its chromo shadow domain to bind to a second region of the histone H3 tail centered around tyrosine 41, and phosphorylation of this residue by nuclear JAK2 blocks this binding (Dawson et al., 2009
). Because the chromo domain and the chromo shadow domain are linked in the same polypeptide, the simultaneous interaction with these two regions of the histone H3 tail would be expected to cooperatively increase HP1α binding avidity. Of note, HP1α also interacts with SUV39H1 (Yamamoto and Sonoda, 2003
) and SETDB1 (Verschure et al., 2005
), which are H3K9 methylases. SUV39H1 methyltransferase activity is required for the spreading of heterochromatin and the recruitment of HP1α. On a nucleosome lacking H3K9 methylation and H3Y41 phosphorylation, HP1α might initially bind through its chromo shadow domain to the histone H3 tail near tyrosine 41, thereby recruiting SUV39H1/SETDB1 to methylate lysine 9 and facilitate HP1α binding through its chromo domain. The 9p24 amplicon appears to engage both JAK2 signaling and JMJD2C to decrease HP1α deposition genome-wide, thereby promoting an active chromatin configuration surrounding functionally critical genes, such as MYC
. JAK2-mediated H3Y41 phosphorylation sets up several positive feedback loops by targeting JMJD2C
itself, as well as IL4R
, which encodes IL4Rα, a subunit of the IL-13 receptor.
Model of cooperative modulation of the cancer epigenome by JAK2 and JMJD2C
Our findings have several implications for the development of new therapeutic modalities for PMBL and HL. Despite the fact that current chemotherapy regimens for HL are quite effective, they fail to cure roughly 20% of patients with advanced stage HL (Diehl et al., 2003
) and 25% of patients with PMBL (Zinzani et al., 2009
). Moreover, PMBL and HL tumors in the mediastinum are often irradiated, causing later sequelae such as coronary artery disease. Inhibitors of JAK2 signaling are just entering the clinic and are beginning to show activity in myelofibrosis associated with activating JAK2 mutations (Santos et al., 2010
). The JAK2 pathway is an attractive therapeutic target in PMBL and HL based on the genetic and functional evidence in the present study along with previous work implicating SOCS1 inactivation in PMBL and HL (Melzner et al., 2005
; Mestre et al., 2005
; Mottok et al., 2009
; Weniger et al., 2006
) and autocrine IL-13 signaling in HL (Skinnider et al., 2002
; Skinnider et al., 2001
). Together, these considerations support the further development of JAK2 inhibitors as potential therapeutic agents in these lymphomas.
Because of the functional redundancy between JAK2 and JMJD2C in some lymphomas with the 9p24 amplicon, it is likely that successful therapy of some cases might require simultaneous inhibition of both enzymes. For example, some HL lines (KM-H2 and L540) showed little or no response to JAK2 inhibition or JMJD2C inhibition as single interventions, but were killed when JAK2 and JMJD2C were simultaneously inhibited. JMJD2C is a potentially druggable enzyme that is an attractive therapeutic target because of its involvement in PMBL and HL. Moreover, JMJD2C is a potentially interesting target in other cancers such as esophageal carcinoma, which can amplify JMJD2C
and depend upon JMJD2C for proliferation (Cloos et al., 2006
; Yang et al., 2000
), and prostate cancer, which can rely upon JMJD2C for androgen-dependent proliferation (Wissmann et al., 2007
). It is important to emphasize that JMJD2C is not required by all cells for proliferation and survival, potentially opening a therapeutic window for cancer treatment. The development of JMJD2C-directed therapeutics may be especially attractive in PMBL and HL as they may have cooperative activity with JAK2-directed agents that are already in clinical trials.