Targeting the Mammalian Mitochondrial Clpp (mClpP) As a Novel Therapeutic Strategy for Acute Myeloid Leukemia

Abstract 3603

The mammalian mitochondrial caseinolytic protease (mClpP) is a nuclear-encoded enzyme complex responsible for degrading excess proteins in the mitochondria. It is functionally similar to the proteasome complex in the cytoplasm and structurally homologous to the bacterial ClpP enzyme complex. While the proteasome has been well characterized as a therapeutic target for the treatment of hematologic malignancy, little is known regarding mClpP as a potential therapeutic target in malignant cells. Therefore, we evaluated the expression of mClpP in acute myeloid leukemia (AML) and normal hematopoietic cells by immunoblotting. Strikingly, mClpP was robustly expressed in AML cell lines (n=3/4) and primary AML patients (n=4/4), but was undetectable in normal bone marrow samples (n = 3) and G-CSF-mobilized peripheral blood mononuclear cells from consenting volunteers donating stem cells for allotransplant (n = 5). We also demonstrated over-expression of mClpP mRNA in these AML samples compared to normal by Q-RT-PCR. Next, we knocked down mClpP and its regulatory chaperone protein mClpX in OCI-AML2 and TEX human leukemia cells using 3 independent shRNA in lentiviral vectors. Target knockdown was confirmed by Q-RT-PCR and immunoblotting. Compared to cells infected with control sequences, knockdown of mClpP reduced the growth and viability of these leukemia cells by > 90%. Knockdown of the chaperone protein mClpX also reduced the growth and viability of these cells, but with less potency than mClpP knockdown.

Rho-zero cells have been depleted of mitochondrial DNA by treatment with ethidium bromide and therefore lack mitochondrial protein synthesis. Through feedback mechanisms, expression of nuclear encoded proteins that contribute to oxidative metabolism are also reduced. Thus, these cells have lower rates of mitochondrial protein accumulation and turnover. We demonstrated that wild type and rho-zero 143B rhabdomyosarcoma cells both express mClpP protein, but levels were slightly lower in the rho-zero cells. We then tested the effects of mClpP knockdown in rho-zero cells and demonstrated that knockdown of mClpP reduced the growth and viability of wild type 143B cells, but had little effect on their rho-zero counterparts.

As a chemical approach to evaluate the effects of mClpP inhibition on AML and normal hematopoietic cells, we synthesized a derivative of recently reported beta-lactone bacterial ClpP inhibitor and generated (3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one that we termed A2–32–01. We confirmed that A2–32–01 inhibited the enzymatic activity of recombinant bacterial ClpP similar to the activity of the reported beta-lactone inhibitors. A2–32–01 induced cell death in TEX, OCI-AML2, and K562 leukemia cells that express mClpP as measured by trypan blue staining. We also isolated mitochondria from these cells after treatment with A2–32–01 and demonstrated that the compound reduced the enzymatic activity of the mClpP protease as measured by cleavage of the fluorogenic substance N-succinyl-Leu-Tyr-7-amidomethylcoumarin (Suc-LY-AMC). In contrast, A2–32–01 was not cytotoxic to HL60 cells that had undetectable mClpP. Likewise, A2–32–01 induced death in 143B wild type cells, but not the rho-zero counterparts.

Finally, we evaluated the effects of mClpP inhibition on primary AML and normal hematopoietic cells. Primary AML and normal hematopoietic cells were treated with increasing concentrations of A2–32–01 and cell viability was measured after 48 hours incubation by Annexin V/PI staining followed by flow cytometry. A2–32–01 did not kill normal hematopoietic cells (n = 3 samples), but was cytotoxic to 5/6 tested primary AML cells.

In summary, we have used genetic and chemical approaches to highlight the mitochondrial protease, mClpP, as a novel therapeutic target for AML.