Hiroshi Kajita

A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo

A pyrrolo-pyrimidine kinase inhibitor, RK-20449, eliminates chemotherapy-resistant human primary AML stem cells in vivo. Taking AML Head-On Like the mythic Lernaean Hydra, acute myeloid leukemia (AML) is hard to kill. It seems like every time one head is cut off, another two—meaner—grow back in its place. Leukemia stem cells (LSCs) are thought to contribute to this resilience; they may survive conventional chemotherapy and increase the risk of relapse. However, it has been difficult to specifically target these cells without also hitting the normal hematopoietic stem cells (HSCs) required for maintaining healthy blood cells. Now, Saito et al. find a new candidate drug that can specifically target LSCs. The authors performed a chemical library screen to target hematopoietic cell kinase (HCK), which they had previously found to be differentially expressed in human LSCs compared with HSCs. They found a candidate HCK inhibitor, RK-20449, which is a pyrrolo-pyrimidine derivative that could bind the active pocket of HCK. In a mouse xenograft of aggressive human AML, RK-20449 greatly reduced LSC burden. If these studies hold true in patients, RK-20449 could accomplish the Herculean task of decreasing the risk of relapse in AML. Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rgnull mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

Overcoming mutational complexity in acute myeloid leukemia by inhibition of critical pathways.

Targeting leukemogenic mutations identified by functional single-cell genomics eliminated human AML in vivo. The right treatments for the right mutations A variety of mutations have been observed in cancer cells from patients with acute myeloid leukemia, but it can be difficult to know which of these mutations contribute to tumorigenesis and should therefore be targeted. To address this issue, Saito et al. isolated subpopulations of leukemic cells with specific mutations and monitored their leukemogenic capacity in immunosuppressed mice. By combining this approach with genomic analysis, the authors were able to identify mutations that drive the evolution of leukemia and figure out effective approaches to target them. Numerous variant alleles are associated with human acute myeloid leukemia (AML). However, the same variants are also found in individuals with no hematological disease, making their functional relevance obscure. Through NOD.Cg-PrkdcscidIl2rgtmlWjl/Sz (NSG) xenotransplantation, we functionally identified preleukemic and leukemic stem cell populations present in FMS-like tyrosine kinase 3 internal tandem duplication–positive (FLT3-ITD)+ AML patient samples. By single-cell DNA sequencing, we identified clonal structures and linked mutations with in vivo fates, distinguishing mutations permissive of nonmalignant multilineage hematopoiesis from leukemogenic mutations. Although multiple somatic mutations coexisted at the single-cell level, inhibition of the mutation strongly associated with preleukemic to leukemic stem cell transition eliminated AML in vivo. Moreover, concurrent inhibition of BCL-2 (B cell lymphoma 2) uncovered a critical dependence of resistant AML cells on antiapoptotic pathways. Co-inhibition of pathways critical for oncogenesis and survival may be an effective strategy that overcomes genetic diversity in human malignancies. This approach incorporating single-cell genomics with the NSG patient-derived xenograft model may serve as a broadly applicable resource for precision target identification and drug discovery.