PNC_¯27 Targeting Plasma Membrane HDM2: A Potentially Novel Therapeutic Approach for Acute Myeloid Leukemia (AML)

Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy. The vast majority of AML patients are immediately refractory to induction therapy or subsequently relapses following transient complete remission, due to persistence of treatment-resistant leukemia stem cells (LSC). Thus, there is a clear need to identify new targets and develop novel and effective therapeutic approaches in this population. HDM2, an E3 ubiquitin-protein ligase, binds to p53 tumor suppressor in the cytoplasm, repressing p53 transcriptional activity and facilitating its proteasomal degradation. Overexpression of HDM2 plays a key role in tumorigenesis. Recently, it has been reported that HDM2 is not only localized to the cytoplasm but can also be found highly expressed on the plasma membrane of various cancer cells (i.e., colon, breast, pancreas) but not on normal counterparts (PNAS 2010 :107(5):1918-23). PNC-27(Oncolyze, Inc.), a 32-amino acid synthetic peptide with a penetratin domain and a HDM-2-binding domain of p53, can bind with cell membrane HDM2, causing membrane pore formation, thus inducing necrosis in solid tumor cells. However, the activity of PNC-27 in AML has not been established. Using flow cytometry analysis and confocal microscopy with anti-HDM2 antibody (Santa Cruz,N-20), we showed that HDM2 was expressed on the membrane of LSC-enriched CD34+CD38- primary blast populations procured from consecutive AML patients (n=6) at levels greater than those observed on normal CD34+CD38- cells [hematopoietic stem cells (HSC)] from healthy individuals (n=12) [median fluorescence intensity (MFI) (HDM-2/IgG control) 2.5±0.6 vs 1.1±0.1; p<0.01)]. Similar results were also observed in AML cell lines (i.e., THP-1, MV4-11,MOLM-13, NOMO1).Further, we showed that exposure to PNC-27 (50-200ug/ml) for 72 hours resulted in significantly reduced viability and colony formation activity (P<0.001) in both AML cell lines and human AML CD34+ cells compared with normal CD34+ cells, in a dose- and time-dependent manner. PNC-27 antileukemia activity was significantly and directly correlated with cell membrane HDM2 expression levels (p<0.05), more HDM2 expression, higher PNC-27 efficacy. Confocal microscopy using fluorescent anti-HDM2 and anti-PNC-27 antibodies showed that PNC-27 and HDM-2 co-localized to the membrane of primary AML CD34+ blasts. Consistent with the previous studies, we also observed membrane pore formation by electron microscopy and cell necrosis by increased LDH levels rather than apoptosis(no change in Annexin V+ cell percentage and pro-apoptosis protein levels) in PNC-27 treated cells.

We further evaluated the efficacy of PNC-27 in vivo . First, CD45.2 BM cells from a MLL^PTD/WTFLT3^ITD/ITD AML mouse model were transplanted into CD45.1 congenic recipient mice. After confirming AML development, mice were treated with PNC-27 (40mg/kg/day; n= 7) or vehicle (n=9) for two weeks. BM cells from the treated mice were transplanted into 2^nd recipient mice (2×10⁶ cells/mouse). A significantly longer survival was observed in the 2^nd BM recipients from PNC-27-treated donors (n=8) compared to the 2^nd BM recipients from vehicle-treated donors (n=8) (median survival: 41 days vs 32 days; p<0.01). Next, human AML cells were transplanted into NSG-SGM3 mice. Upon confirming >10% human CD45+ cell engraftment in periphery blood (PB), the mice were treated with PNC-27 (40mg/kg/day; n=10) or vehicle (n=10) for 2 weeks, and BM cells (2×10⁶ cells/mouse) from the treated mice were transplanted into 2^nd recipient mice. Significantly reduced human CD45+ engraftment in PB at 12 weeks (59.2±7.4 vs 94.6±0.94, p<0.05) and prolonged survival were observed in the 2^nd BM recipients from PNC-27-treated donors (n=10) compared to the 2^nd BM recipients from vehicle-treated donors (n=7). After four months follow-up, all recipients receiving BM cells from PNC-27-treated donors survived while all recipients receiving BM cells from vehicle-treated donors died (median survival was "not reached" vs 93 days, p<0.0001). Of note wild-type B6 mice treated with PNC-27 (80mg/kg) or vehicle for 2 weeks showed no difference on WBC counts and BM cell counts, suggesting no toxicity on normal hematopoiesis.

In conclusion, our results suggest PNC-27 effectively targets AML LSC-enriched population by binding with cell membrane HDM2 to induce necrosis, providing a rationale for a rapid translation of this drug into the clinic.