DNA-damaging chemotherapeutic regimens are clinically efficacious for the KSHV-associated malignancies KS and PEL. Typically, the tumor response to chemotherapy is dependent upon functional p53. However, an in-depth analysis of p53 function in PEL has been missing, because multiple proteins of the PEL-associated human tumor virus KSHV were shown to inhibit p53 in ectopic expression experiments (27
), thus asserting the dogma that all DNA tumor viruses inactivate p53. If this were true, the presence of KSHV would severely limit the treatment options for PEL and other KSHV-associated cancers. PEL should not be responsive to CHOP, which is in contrast to the clinical experience (64
). To test the hypothesis that p53 is fully functional in PEL despite the presence of viral oncogenes, we assessed p53 status and function in response to the prototypical DNA-damaging agent doxorubicin. Analyzing the most extensive panel of PEL lines published to date, we find that, similar to KS (38
), p53 mutations are rarely detected in PEL (Table ). DNA damage-induced p53 activation is intact in PEL harboring wild-type p53, as evidenced by target gene induction and cell cycle arrest following treatment with clinically relevant doses of doxorubicin (Fig. , , and ). These data imply that in PEL the p53 status dictates the response to chemotherapeutics regardless of KSHV viral latent proteins.
We corroborated our initial observations by modifying p53 signaling using the well-characterized activator Nutlin-3 (Fig. ) or the p53 inhibitor pifithrin-alpha (Fig. ). The phenotypic response to each of these drugs, as well as the p53-dependent transcriptional response, correlated tightly with p53 status, adducing that DNA damage-induced p53 signaling is functional. How, then, can we reconcile these data with reports demonstrating disruption of p53 signaling by lytic and latent KSHV viral proteins? KSHV lytic proteins have been shown to inhibit p53 function through coactivator sequestration (Rta/ORF50) (18
), repression of ATM-mediated DNA damage signaling (v-IRF) (62
), or other unknown mechanisms (K-bZIP) (48
). Neither Rta/ORF50, K-bZIP, nor vIRF-1 is expressed during latency in PEL (26
). These transcripts are only detectable upon viral reactivation. Herpesvirus reactivation leads to replication of the viral genomic DNA, which is resolved through recombination into unit-length pieces that are packed into the virion. This process generates many DNA double-strand ends, and it is therefore highly plausible that KSHV evolved to inhibit the p53/ATM response during the lytic phase of the viral life cycle.
The KSHV latency-associated nuclear antigen (LANA) also binds and inhibits p53 function in reporter assays (27
). Conversely, p53 can inhibit the LANA promoter (37
). Unlike the lytic proteins, LANA is constitutively expressed in all PEL cells (19
). LANA, like p53, is a relatively “sticky” protein, binding to many partners in vitro. Due to the multitude of LANA binding partners (≥10) and functions (63
), it has been considerably more difficult to establish the specificity and functionality of the p53-LANA complex. LANA binds to the histone core components H2A, H2B, and H1, thereby tethering the viral episome to host chromatin (4
). This interaction results in the characteristic nuclear speckled pattern observed using LANA-specific monoclonal antibodies (39
). It is conceivable that some p53 would be sequestered into these complexes, which also contain Ku70, Ku80, and PARP-1. The results presented here show that the nature of the LANA-p53 complex is such that enough functional p53 is available to mediate PEL cell cycle inhibition in response to DNA damage or to Nutlin-3. In fact, upon Nutlin-3 treatment p53 and LANA occupy distinct, nonoverlapping nuclear compartments (Fig. S1 in the supplemental material).
The rarity of p53 mutations observed herein expands upon an earlier report (46
), which similarly did not detect p53 mutations in primary PEL biopsies. The p53 mutations in two of the cell lines examined (Table ) may reflect the origins of the patient samples. BCP-1 cells (p53, S262/S262) were derived from an HIV-negative, 94-year-old man with previous history of both KS and colon cancer (5
). This individual was treated for 3 years with prolonged chemotherapy before succumbing to the disease, whereupon the BCP-1 cell line was isolated. Likewise, BCBL-1 (wild type/M246I) cells were derived from an HIV-positive patient who underwent prior chemotherapeutic treatment with doxorubicin (42
). Unlike other primary effusions, which typically fail to grow after a period of 14 days upon explantation in culture, BCBL-1 cells grew rapidly with no discernible lag phase (B. Herndier and D. Ganem, personal communication). BCLM and JSC-1 were isolated prior to therapeutic administration and maintained wild-type p53 function. Thus, long-term PEL culture is compatible with p53 activity, and only prolonged treatment with DNA-damaging agents in vivo appears to select for p53 mutations.
The M246I and S262 mutations identified in these PEL lines were previously reported in other cancers. The M246I mutation (BCBL-1) was initially identified in the H23 lung cancer cell line and was reported to maintain DNA binding to consensus p53 binding elements (56
). However, transactivation of nonconsensus elements by p53 M246I was limited, resulting in decreased apoptotic induction. While the Mdm2 promoter (consensus element) was effectively induced by M246I, PIG3 (nonconsensus) transactivation was significantly reduced. This is consistent with the phenotype of BCBL-1 cells, which demonstrated intense induction of p53 target genes following Nutlin-3 treatment yet failed to undergo significant apoptosis (Fig. ). Far less is known about the p53 S262 insertion mutant. Previously identified in pancreatic cancer (11
), a characterization of the mutant has yet to be conducted. Our findings suggest that S262 has little transactivation potential, as few genes were induced in the Nutlin-3 array studies. Additionally, BCP-1 cells (S262/S262) demonstrated a low background level of all p53 transcripts assayed, further implying that this signaling pathway is disrupted (data not shown). Understanding how individual p53 mutations contribute to therapeutic response will be essential to providing individualized PEL therapy.
Structural studies of the p53-Mdm2 interaction have revealed evidence that three key residues (Phe, Trp, and Lev) of p53 bind to a deep cavity on the Mdm2 surface (44
). Nutlins represent the most potent and selective inhibitors of this interaction described to date. Nutlins are cis
-imidizoline analogs, capable of penetrating cellular membranes, and thus can be administered orally (68
). Nutlin-3 was well tolerated in murine xenograph studies (up to 200 mg/g of body weight twice daily), wherein the growth of p53-positive tumors was inhibited (67
). Recent studies implicate Nutlin-3 as a novel therapeutic for B-cell chronic lymphocytic leukemia (B-CLL) and acute myeloid leukemia (AML) (16
). Interestingly, these previous studies utilized the purified, active Nutlin-3a enantiomer. Herein, we studied the effects of the racemic mixture (containing a 1:1 ratio of active [3a] versus inactive [3b] enantiomer). By comparison, PELs were more sensitive to Nutlin-3 treatment than B-CLL and AML (Fig. ; 2.5 to 5.0 μM racemic versus 4.7 and 5 μM Nutlin-3a, respectively), perhaps reflecting their phenotypically elevated Hdm2 expression. PELs harboring the wild-type p53 allele exhibited significant apoptosis following Nutlin-3 treatment; however, PEL lines with homozygous, mutant p53 failed to proliferate only at increased doses of drug (Fig. ). BC-3 cells deviated somewhat from the phenotype of other p53 wild-type cell lines, and further studies are under way to clarify this phenotype. One possible explanation is that BC-3 cells express a high level of HdmX (unpublished observation). Increased HdmX levels would explain their partial resistance to Nutlin-3, since Nutlin-3 fails to block the p53-HdmX interaction (50
). Alternatively, Nutlin-3 may affect other targets than p53 (25
). A key finding of this study is that Hdm2 is overexpressed in PEL. Moreover, Hdm2 mRNA levels can be utilized to classify PEL away from diffuse large B-cell lymphoma and other B-cell-lymphoproliferative diseases (Fig. ). The underlying mechanism for this phenotype is currently under investigation. Regardless, these data explain the dramatic susceptibility of this tumor type to Nutlin-3.
The use of a novel targeted p53 real-time QPCR array allowed us to molecularly characterize PEL response to Nutlin-3. Activation of the p53 transcriptome following Nutlin-3 addition differs between PEL and other hematological malignancies. In B-CLL, Nutlin-3a activates Mdm2, p21, and PUMA expression but not BAX (16
). In AML, Nutlin-3a activates Mdm2, p21, and NOXA but not PUMA or BAX (41
). Here we find that in PEL, Nutlin-3 rapidly (<8 h) activates multiple p53 targets, including p21, NOXA, PUMA, and BAX (Fig. ). Independent of the specific transcriptional targets activated, the cellular outcome, namely growth inhibition and apoptosis, is the same for PEL, B-CLL, and AML. PEL is a rapidly progressing disease, wherein the immunocompromised status of the patient often influences the physician's ability to administer therapy. Our findings suggest that determination of p53 and Hdm2 status is critical to the assessment of potential therapeutic regimens. KSHV oncogenes did not influence the ability of PEL to respond to DNA-damaging agents. However, they may modulate p53 function at steady-state growth or during KSHV lytic replication. Nutlins and other p53-activating compounds may prove highly efficacious for the treatment of PEL patients.