CLT030, a leukemic stem cell-targeting CLL1 antibody-drug conjugate for treatment of acute myeloid leukemia.

The current standard of care for acute myeloid leukemia (AML) is largely ineffective with very high relapse rates and low survival rates, mostly due to the inability to eliminate a rare population of leukemic stem cells (LSCs) that initiate tumor growth and are resistant to standard chemotherapy. RNA-sequencing analysis on isolated LSCs confirmed C-type lectin domain family 12 member A (CLL1, also known as CLEC12A) to be highly expressed on LSCs but not on normal hematopoietic stem cells (HSCs) or other healthy organ tissues. Expression of CLL1 was consistent across different types of AML. We developed CLT030 (CLL1-ADC), an antibody-drug conjugate (ADC) based on a humanized anti-CLL1 antibody with 2 engineered cysteine residues linked covalently via a cleavable linker to a highly potent DNA-binding payload, thus resulting in a site-specific and homogenous ADC product. The ADC is designed to be stable in the bloodstream and to release its DNA-binding payload only after the ADC binds to CLL1-expressing tumor cells, is internalized, and the linker is cleaved in the lysosomal compartment. CLL1-ADC inhibits in vitro LSC colony formation and demonstrates robust in vivo efficacy in AML cell tumor models and tumor growth inhibition in the AML patient-derived xenograft model. CLL1-ADC demonstrated a reduced effect on differentiation of healthy normal human CD34+ cells to various lineages as observed in an in vitro colony formation assay and in an in vivo xenotransplantation model as compared with CD33-ADC. These results demonstrate that CLL1-ADC could be an effective ADC therapeutic for the treatment of AML.

Phosphorylation of Dishevelled by protein kinase RIPK4 regulates Wnt signaling.

Receptor-interacting protein kinase 4 (RIPK4) is required for epidermal differentiation and is mutated in Bartsocas-Papas syndrome. RIPK4 binds to protein kinase C, but its signaling mechanisms are largely unknown. Ectopic RIPK4, but not catalytically inactive or Bartsocas-Papas RIPK4 mutants, induced accumulation of cytosolic ß-catenin and a transcriptional program similar to that caused by Wnt3a. In Xenopus embryos, Ripk4 synergized with coexpressed Xwnt8, whereas Ripk4 morpholinos or catalytic inactive Ripk4 antagonized Wnt signaling. RIPK4 interacted constitutively with the adaptor protein DVL2 and, after Wnt3a stimulation, with the co-receptor LRP6. Phosphorylation of DVL2 by RIPK4 favored canonical Wnt signaling. Wnt-dependent growth of xenografted human tumor cells was suppressed by RIPK4 knockdown, suggesting that RIPK4 overexpression may contribute to the growth of certain tumor types.

Mammary tumor regression elicited by Wnt signaling inhibitor requires IGFBP5.

Wnt ligand-driven tumor growth is inhibited by the soluble Wnt inhibitor Fzd8CRD, but the mechanism through which this effect is mediated is unknown. In the MMTV-Wnt1 mouse model, regression of mammary tumors by Fzd8CRD treatment coincides with an acute and strong induction of insulin-like growth factor (IGF)-binding protein IGFBP5, an antagonist of IGF signaling that mediates involution of mammary gland in females after offspring are weaned. In this study, we show that repression of this IGF inhibitory pathway is crucial for Wnt-driven growth of mammary tumors. We found that IGFBP5 regulation was mediated by the ß-catenin-dependent Wnt pathway. Wnt, in addition to IGF ligands, facilitated tumor growth by paracrine communication among tumor cells. In addition, Fzd8CRD caused precocious induction of IGFBP5 in normal mammary glands undergoing involution, implying an acceleration of the involution process by inhibition of Wnt signaling. The molecular and phenotypic parallel between tumor regression and mammary gland involution suggests that Wnt-driven mammary tumors use the same growth mechanism as proliferating normal mammary glands.

Wnt isoform-specific interactions with coreceptor specify inhibition or potentiation of signaling by LRP6 antibodies.

ß-Catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.

Ubiquitin hydrolase Dub3 promotes oncogenic transformation by stabilizing Cdc25A.

The dual specificity (Tyr/Thr) phosphatase Cdc25A activates cyclin-dependent kinases (Cdks) to promote cell-cycle progression and has significant oncogenic potential. Cdc25A protein levels are regulated tightly in normal tissues, but many human cancers overexpress Cdc25A. The underlying mechanism for overexpression has been enigmatic. Here we show that Cdc25A is stabilized by the ubiquitin hydrolase Dub3. Upon binding Cdc25A, Dub3 removes the polyubiquitin modifications that mark Cdc25A for proteasomal degradation. Dub3 knockdown in cells increased Cdc25A ubiquitylation and degradation, resulting in reduced Cdk/Cyclin activity and arrest at G1/S and G2/M phases of the cell cycle. In contrast, acute Dub3 overexpression produced a signature response to oncogene induction: cells accumulated in S and G2 because of replication stress, and activated a DNA damage response. Dub3 also transformed NIH-3T3 cells and cooperated with activated H-Ras to promote growth in soft agar. Importantly, we show that Dub3 overexpression is responsible for an abnormally high level of Cdc25A in a subset of human breast cancers. Moreover, Dub3 knockdown significantly retarded the growth of breast tumour xenografts in nude mice. As a major regulator of Cdc25A, Dub3 is an example of a transforming ubiquitin hydrolase that subverts a key component of the cell cycle machinery.

Stromal cell-derived factor-1/CXCL12 contributes to MMTV-Wnt1 tumor growth involving Gr1+CD11b+ cells.

BACKGROUND: Histological examinations of MMTV-Wnt1 tumors reveal drastic differences in the tumor vasculature when compared to MMTV-Her2 tumors. However, these differences have not been formally described, nor have any angiogenic factors been implicated to be involved in the Wnt1 tumors. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that MMTV-Wnt1 tumors were more vascularized than MMTV-Her2 tumors, and this correlated with significantly higher expression of a CXC chemokine, stromal cell-derived factor-1 (SDF1/CXCL12) but not with VEGFA. Isolation of various cell types from Wnt1 tumors revealed that SDF1 was produced by both tumor myoepithelial cells and stromal cells, whereas Her2 tumors lacked myoepithelial cells and contained significantly less stroma. The growth of Wnt1 tumors, but not Her2 tumors, was inhibited by a neutralizing antibody to SDF1, but not by neutralization of VEGFA. Anti-SDF1 treatment decreased the proportion of infiltrating Gr1(+) myeloid cells in the Wnt1 tumors, which correlated with a decrease in the percentage of endothelial cells. The involvement of Gr1(+) cells was evident from the retardation of Wnt1 tumor growth following in vivo depletion of these cells with an anti-Gr1-specific antibody. This degree of inhibition on Wnt1 tumor growth was comparable, but not additive, to the effect observed with anti-SDF1, indicative of overlapping mechanisms of inhibition. In contrast, Her2 tumors were not affected by the depletion of Gr1(+) cells. CONCLUSIONS/SIGNIFICANCE: We demonstrated that SDF1 is important for Wnt1, but not for HER2, in inducing murine mammary tumor and the role of SDF1 in tumorigenesis involves Gr1(+) myeloid cells to facilitate growth and/or angiogenesis.

Wnt signaling induces differentiation of progenitor cells in organotypic keratinocyte cultures.

BACKGROUND: Interfollicular skin develops normally only when the activity of the progenitor cells in the basal layer is counterbalanced by the exit of cells into the suprabasal layers, where they differentiate and cornify to establish barrier function. Distinct stem and progenitor compartments have been demonstrated in hair follicles and sebaceous glands, but there are few data to describe the control of interfollicular progenitor cell activity. Wnt signaling has been shown to be an important growth-inducer of stem cell compartments in skin and many other tissues. RESULTS: Here, we test the effect of ectopic Wnt1 expression on the behavior of interfollicular progenitor cells in an organotypic culture model, and find that Wnt1 signaling inhibits their growth and promotes terminal differentiation. CONCLUSION: These results are consistent with the phenotypes reported for transgenic mice engineered to have gain or loss of function of Wnt signaling in skin, which would recommend our culture model as an accurate one for molecular analysis. Since it is known that canonical ligands are expressed in skin, it is likely that this pathway normally regulates the balance of growth and differentiation, and suggests it could be important to pathogenesis.

The transforming activity of Wnt effectors correlates with their ability to induce the accumulation of mammary progenitor cells.

Ectopic activation of the Wnt signaling pathway is highly oncogenic for many human tissues. Here, we show that ectopic Wnt signaling increases the effective stem cell activity in mouse mammary glands in vivo. Furthermore, Wnt effectors induce the accumulation of mouse mammary epithelial progenitors (assayed by Hoechst dye exclusion, a surrogate stem cell marker, side population cells) both in vivo and in vitro. The longevity of stem cells makes them good candidate tumor precursors, and we propose that Wnt-induced progenitor amplification is likely to be key to tumor initiation. In support of this notion, mammary glands from a tumor-resistant strain of mice (carrying a null mutation in syndecan-1) contain fewer side population cells. When this strain is crossed to mice that overexpress effectors of the beta-catenin/T cell factor Wnt pathway, the amplification of progenitors is reduced, together with all subsequent events of tumor development. We propose that the growth dynamic of the stem cell fraction is a major determinant of tumor susceptibility.

Mammary gland development requires syndecan-1 to create a beta-catenin/TCF-responsive mammary epithelial subpopulation.

Mice with a null mutation in the cell surface heparan sulfate (HS) proteoglycan, syndecan-1 (Sdc1), develop almost normally, but resist mammary tumor development in response to Wnt-1. Here, we test the hypothesis that Sdc1 promotes Wnt-1-induced tumor development by interacting with the Wnt cell surface signaling complex. Thus, the response of Sdc1-/- mammary epithelial cells (mecs) to the intracellular, activated Wnt signal transducer, DeltaNbeta-catenin, was assayed both in vitro and in vivo, to test whether beta-catenin/TCF transactivation was Sdc1-independent. Surprisingly, we found that the expression of a canonical Wnt pathway reporter, TOP-FLASH, was reduced by 50% in both unstimulated Sdc1-/- mecs and in stimulated cells responding to Wnt1 or DeltaNbeta-catenin. Tumor development in response to DeltaNbeta-catenin was also significantly delayed on a Sdc1-/- background. Furthermore, the average beta-catenin/TCF transactivation per cell was normal in Sdc1-/- mec cultures, but the number of responsive cells was reduced by 50%. Sdc1-/- mecs show compensatory changes that maintain the number of HS chains, hence these experiments cannot test the coreceptor activity of HS for Wnt signaling. We propose that TCF-dependent transactivational activity is suppressed in 50% of cells in Sdc1-/- glands, and conclude that the major effect of Sdc1 does not map to the activity of the Wnt signaling complex, but to another pathway to create or stabilize the beta-catenin/TCF-responsive tumor precursor cells in mouse mammary gland.