Acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are cancers of the bone marrow that in adults, occur mainly in the elderly. About 19,000 Americans develop these diseases annually and the 5 year survival rate is 30-40%. The prognosis for elderly patients is even worse with a 5 year survival rate of 10%. Treatment for AML and ALL involves induction chemotherapy using the deoxycytidine analog cytarabine followed by consolidation therapy or stem cell transplant. The major obstacle to long-term disease-free survival is relapse after treatment due to chemoresistance. Recurrent disease is much more difficult to treat successfully and elderly patients often have trouble tolerating repeated chemotherapy treatments. Chemoresistance can occur by several mechanisms including reduced activation of cytarabine via downregulated deoxycytidine kinase (dCK) levels or decreased uptake via lowered Equilibrative Nucleoside Transporter 1 (ENT-1) levels. Low expression of both dCK and ENT-1 correlate with poor prognosis in patients with acute leukemias.
Another contributor to cytarabine resistance is the interaction of leukemia cells with bone marrow stroma. This interaction confers protection to leukemia cells from cytarabine by inhibiting apoptosis and ENT-1 function. Additionally, activation of NF-κβ by protein kinase C (PKC) promotes proliferation and inhibits apoptosis in AML. Increased expression of PKC isoforms α, β1, and β2 have been detected in both AML and ALL and promote leukemic proliferation and survival by activation of ERK and the anti-apoptotic protein BCL-2.
The development of novel therapeutic agents or modifying existing drugs may be able to overcome chemoresistance in AML and ALL and improve clinical outcomes. KPC34 is a novel phospholipid conjugate of the deoxycytidine analog gemcitabine. Gemcitabine acts similarly to cytarabine and is administered IV to treat various tumors and lymphomas, but is rarely used for leukemias. Compared to cytarabine, gemcitabine treatment inhibits DNA replication more effectively, prevents repair of damaged DNA, and uniquely inhibits ribonucleotide reductase. KPC34's novel properties result in improved pharmacokinetics compared to gemcitabine and may be able to overcome leukemic chemoresistance to cytarabine by bypassing the need for ENT-1 uptake and dCK activation. In addition, KPC34 can be administered orally, is predicted to cross the blood-brain barrier, allowing it to target CNS-infiltrating leukemias. Finally, KPC34’s lipid moiety is predicted to inhibit the activity of PKC.
In order to determine if KPC34 has activity against acute leukemias we tested it against a variety of human and murine leukemic cell lines in vitro. KPC34 was highly cytotoxic to with IC50s in the nM range. The mean IC50 was 45.92 nM (range from 7.39-175.6 nM). Exposure of leukemia cells to KPC34 resulted in the induction of apoptosis as assessed by annexin V and PI assays. Human leukemic cell lines treated with KPC34 had dose-dependent reduction of colony formation, where normal donor hematopoietic stem cells did not. To test the ability of KPC34 to overcome stroma-mediated chemoresistance, SUPB15 cells were incubated in the presence or absence of a bone marrow stroma cell line for 48 hrs. The cells were then treated with 10 nM cytarabine or KPC-34. No significant difference was seen between either treatment without stroma, while KPC34 significantly increased apoptosis in the presence of stroma (p value ≤ 0.001, by Student’s t test).
In order to assess the activity of KPC34 in vivo, we injected C57/BL6 mice with a syngeneic, murine Ph+ pre-B cell ALL, luciferase expressing cell line. When engraftment was established by bioluminescence imaging, mice were treated with 25 mg/kg KPC34 or water by oral gavage on days 1, 4 and 7. Treatment resulted in a significant prolongation of survival with treated mice having a median survival of 26 days compared to 13 days for controls (p value of 0.0042, by log-rank test).
These data demonstrate the activity of KPC34 in multiple preclinical models of leukemia and support its continued translation to the clinic.
Kucera:Wake Forest Baptist Health: KPC 34 Patent, KPC 34 Patent Patents & Royalties.
Asterisk with author names denotes non-ASH members.