Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD-positive and -negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated that re-activation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML.

Background: RhoG is a ubiquitously expressed member of the Rho family of GTPases. Results: RhoG-deficient platelets display severely impaired GPVI-dependent platelet activation. Conclusion: RhoG plays an important role in GPVI-FcR␥ complex-mediated platelet activation and thrombus formation. Significance: Our study enhances the understanding of the molecular mechanisms of GPVI-FcR␥ complex activation.

C o m m e n t a r y2 Divergent functions for Dickkopf-1Dickkopf-1 (DKK-1) was first identified in Xenopus as an extracellular antagonist of canonical Wnt signaling (1), a fundamental pathway that controls cell fate, survival, and proliferation. Aberrant activation of Wnt signaling also drives tumorigenesis in colon, breast, hepatocellular, and many other cancers (2-4). As a Wnt signaling inhibitor, DKK-1 suppresses tumorigenesis in some cancer cells, including renal cell carcinoma (5), breast carcinoma (6), and colon cancer cells (7-9). However, DKK-1 is often elevated in cancers, is associated with aggressive disease, and promotes proliferation in multiple cell types (10, 11), suggesting that DKK-1 also functions independently of Wnt inhibition. In this issue, Kimura and colleagues investigate the molecular events that lead to DKK-1-induced cancer cell proliferation (12). Their work has identified a Wnt-independent signaling role for DKK-1 through interaction with the type-II transmembrane protein cytoskeleton-associated protein 4 (CKAP4; also known as p63, ERGIC-63, or CLIMP-63). These findings also indicate that the DKK-1/CKAP4 interaction represents a promising therapeutic target for cancer.CKAP4 is a DKK-1 receptor that activates AKT signaling DKK family proteins contain two cysteinerich domains (CRDs). The C-terminal CRD (CRD-2) of DKK-1 inhibits Wnt signaling by directly binding to the Wnt coreceptors LRP5/6 (LDL-related proteins 5 and 6), thereby preventing interaction with Wnts, and by binding to the transmembrane protein KREMEN to promote LRP5/6 internalization (11, 13-15). Kimura et al. have now demonstrated that CRD-1 of DKK-1 binds to CKAP4 at the apical surface of epithelial cells (Figure 1 and ref. 12). Furthermore, DKK-1 binds to CKAP4 with high affinity that is comparable to but independent of its interaction with LRP5/6. Upon DKK-1 binding, CKAP4 recruits phosphatidylinositol-3 kinase (PI3K) to its cytoplasmic domain through interaction with the SH3 domain of the p85α subunit of PI3K. Through this mechanism, DKK-1 activates AKT and promotes proliferation. These findings substantially expand our understanding of DKK-1 signaling mechanisms and define a previously unrecognized input to the PI3K/AKT pathway.The findings of Kimura et al. also raise questions about the potential functions of other DKK family members (12). The DKK family comprises four conserved proteins, DKK-1 through -4 (and DKK-like 1, which lacks the CRDs). DKK-1 antagonizes Wnt/β-catenin signaling, activates AKT (as shown here), and may also activate JNK signaling (16); however, the functions of other DKK family members are less well characterized. DKK-2 and -4 antagonize Wnt signaling, similar to DKK-1, but DKK-2 can also activate canonical Wnt signaling depending on the cellular context (11,17). In contrast, DKK-3 positively regulates Wnt signaling (18) and binds to integrin α6b to induce myogenesis in zebrafish (19). As Kimura and colleagues point out (12), CRD-1 is highly conserved among the four Dkk genes, raising the possib...

Wnt signaling plays important roles in stem cell self-renewal and differentiation in adults as well as in embryonic development. Mutations that activate canonical Wnt/β-catenin signaling also initiate and maintain several cancer states, including colorectal cancer and leukemia, and hence Wnt inhibitors are currently being explored as therapeutic options. In this review, we summarize previous studies and update recent findings on canonical Wnt signaling and its components, as well as their roles in somatic stem cell homeostasis and maintenance of cancer initiating cells.

C-PC (C-phycocyanin) is a water-soluble biliprotein from the filamentous cyanobacterium Spirulina platensis with potent antioxidant, anti-inflammatory and anticancerous properties. In the present study, the effect of C-PC was tested on the proliferation of doxorubicin-sensitive (S-HepG2) and -resistant (R-HepG2) HCC (hepatocellular carcinoma) cell lines. These studies indicate a 50% decrease in the proliferation of S- and R-HepG2 cells treated with 40 and 50 microM C-PC for 24 h respectively. C-PC also enhanced the sensitivity of R-HepG2 cells to doxorubicin. R-HepG2 cells treated with C-PC showed typical apoptotic features such as membrane blebbing and DNA fragmentation. Flow-cytometric analysis of R-HepG2 cells treated with 10, 25 and 50 microM C-PC for 24 h showed 18.8, 39.72 and 65.64% cells in sub-G(0)/G(1)-phase respectively. Cytochrome c release, decrease in membrane potential, caspase 3 activation and PARP [poly(ADP-ribose) polymerase] cleavage were observed in C-PC-treated R-HepG2 cells. These studies also showed down-regulation of the anti-apoptotic protein Bcl-2 and up-regulation of the pro-apoptotic Bax (Bcl2-associated X-protein) protein in the R-HepG2 cells treated with C-PC. The present study thus demonstrates that C-PC induces apoptosis in R-HepG2 cells and its potential as an anti-HCC agent.