OPN Maintains Leukemia Stem Cells Survival By Suppressing PUMA

Acute myeloid leukemia (AML) is the most common acute leukemia in adults. In the era of chemotherapy, the cure rate of AML is 35 to 40% of adult patients who are 60 years of age or younger while it is only 5 to 15% of patients who are older than 60 years of age could achieve relative long term survival. Leukemia stem cells (LSCs) population is a small proportion in leukemia cells but plays an important role in initiation of AML and is the main cause of the recurrence after chemotherapy due to the ability of maintaining quiescence and other mechanisms to sustain survival. How to eradicate LSCs was investigated intensively during past years, but how LSCs maintain survival is still elusive. Here we show that osteopontin(OPN), a member of extracellular matrix protein, is essential for AML stem cells survival and could be a novel and potent therapeutic target for eradicating LSCs.

We used RNA interference system to knock down OPN in a mouse model of MLL-AF9 induced AML. We examined the survival of AML mice and found that OPN knockdown (KD) group showed a significant prolongation of survival. Colony forming assay showed that OPN KD AML cells formed colonies in a lower frequency than that of control AML cells. Flow cytometry showed the frequency of LSC in KD AML cells was lower than that in control AML cells(0.04±0.02% vs 3.61±0.32%) and the median fluorescence density of c-Kit is lower in KD AML cells than of control AML cells(759.2±80.8 vs 1353.5±27.5). Brdu assay indicated an increased quiescent fraction in G0/G1 phases (64.9±8.9% vs 53.6±3.4%) of KD AML cells. The apoptosis of KD AML cells is increased than that in the control AML cells(14.82±3.4% vs 4.9±1.2%). Most interestingly, the change of phenotype is much more prominent in the LSCs population, ( comparing KD group and control group, frequency of G0/G1 in LSCs: 50.2±7.5% vs 26.4±3.7%; frequency of apoptotic cells in LSCs: 58.42±5.38% vs 7.45±1.57%). Limiting dilution assay confirmed that lower frequency of LSCs in the KD AML cells than in control(1/1204 vs 1/214). Thus we concluded that OPN plays a very important role in maintain survival of LSCs.

To determine whether OPN knockdown could induce the same phenotype in human AML cells. We constructed OPN knockdown cell-line model by RNA interference system. Human AML cell-lines (SKM-1, ML2, OCI-AML3, SKM-1 FL3-ITD knock in) presented the same phenotype such as the increase of apoptosis and the decrease of the fraction of cells entering cell cycle. Then we performed Realtime PCR and western blot in mouse model to reveal the underlying mechanisms. Realtime PCR showed a significantly up-regulation of P16, P18, P21, which is related to the cell cycle arrest; western blot showed a significantly increase of PUMA and cleaved-caspase3, which indicated that knockdown OPN could promote apoptosis through repress the function of BCL2 by up-regulate PUMA in protein level. Interestingly, incubation of recombinant OPN protein with OPN KD cells did not rescue the phenotype suggesting that intracellular function of OPN in LSCs is important for its survival. The detailed mechanism of how OPN regulate PUMA is still under investigation.

In our current study, it showed that OPN knock down can significantly prolong the survival of leukemic mice and decrease the leukemia burden by decreasing the stemness of LSC and promoting LSC apoptosis by inducing PUMA protein function. Thus it could be a good therapeutic target for eradicating LSCs in AML.