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Dysregulated cellular differentiation is a hallmark of acute leukemogenesis. Phosphatases are widely suppressed in cancers but have not been traditionally associated with differentiation. In this study, we found that the silencing of protein phosphatase 2A (PP2A) directly blocks differentiation in acute myeloid leukemia (AML). Gene expression and mass cytometric profiling revealed that PP2A activation modulates cell cycle and transcriptional regulators that program terminal myeloid differentiation. Using a novel pharmacological agent, OSU-2S, in parallel with genetic approaches, we discovered that PP2A enforced c-Myc and p21 dependent terminal differentiation, proliferation arrest, and apoptosis in AML. Finally, we demonstrated that PP2A activation decreased leukemia-initiating stem cells, increased leukemic blast maturation, and improved overall survival in murine Tet2−/−Flt3ITD/WT and human cell-line derived xenograft AML models in vivo. Our findings identify the PP2A/c-Myc/p21 axis as a critical regulator of the differentiation/proliferation switch in AML that can be therapeutically targeted in malignancies with dysregulated maturation fate.

Antibody-drug conjugates directed against tumor-specific targets have allowed targeted delivery of highly potent chemotherapy to malignant cells while sparing normal cells. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal protein with limited expression on normal adult tissues and is overexpressed on the surface of malignant cells in mantle cell lymphoma, acute lymphocytic leukemia with t(1;19)(q23;p13) translocation, and chronic lymphocytic leukemia. This differential expression makes ROR1 an attractive target for antibody-drug conjugate therapy, especially in malignancies such as mantle cell lymphoma and acute lymphocytic leukemia, in which systemic chemotherapy remains the gold standard. Several preclinical and phase 1 clinical studies have established the safety and effectiveness of anti-ROR1 monoclonal antibody–based therapies. Herein we describe a humanized, first-in-class anti-ROR1 antibody-drug conjugate, huXBR1-402-G5-PNU, which links a novel anti-ROR1 antibody (huXBR1-402) to a highly potent anthracycline derivative (PNU). We found that huXBR1-402-G5-PNU is cytotoxic to proliferating ROR1+ malignant cells in vitro and suppressed leukemia proliferation and extended survival in multiple models of mice engrafted with human ROR1+ leukemia. Lastly, we show that the B-cell lymphoma 2 (BCL2)-dependent cytotoxicity of huXBR1-402-G5-PNU can be leveraged by combined treatment strategies with the BCL2 inhibitor venetoclax. Together, our data present compelling preclinical evidence for the efficacy of huXBR1-402-G5-PNU in treating ROR1+ hematologic malignancies.

Introduction: Sialic acid-binding immunoglobulin-like lectins (Siglec) are a group of lectins that regulate innate and adaptive immune function via glycan recognition. We and others have shown overexpression of Siglec-6, a member of Siglec family, on B cells from patients with chronic lymphocytic leukemia (CLL) compared to normal donor derived B cells. While placental expression of Siglec-6 has been shown to regulate invasion of trophoblast cells by binding to glycodelin, the biochemical role of Siglec-6 in CLL patients is not known. We describe here for the first time the functional relevance of Siglec-6 and its ligand sialyl Tn (sTn) in cell adhesion and migration in CLL. Biochemical mechanisms of Siglec-6 mediated cell adhesion and migration through DOCK8 dependent activation of Cdc42 associated with actin polymerization in CLL cells are presented. Further, the physiological relevance of Siglec-6/ DOCK8 axis in CLL cell adhesion and migration is validated using primary CLL patient samples, and genetically engineered loss of function Siglec-6 and DOCK8 mutant MEC1 CLL cell line. These studies thus elucidate the biological role of Siglec-6 in malignant CLL B cells and demonstrate therapeutic opportunities targeting Siglec-6 in CLL. Methods: Flow cytometry was used to analyze surface expression of Siglec-6 and sTn in CLL patients and normal donors. Transwell migration assay was used to assess in-vitro migratory role of Siglec-6. Mass spectrometry analysis was performed to identify Siglec-6 interacting proteins. CRISPR/Cas9 technique was used to generate knock-out (KO) cell lines for mechanistic studies. Phalloidin staining followed by confocal imaging was used to examine actin polymerization. Cdc42 activation was evaluated using a commercial kit which uses specialized PAK1-PBD agarose beads to pull down GTP-bound Cdc42. To study the in-vivo migratory role of Siglec-6, MEC1 CLL cell line or primary CLL cells were blocked with an isotype antibody or Siglec-6 targeted antibody and injected into the tail vein of NSG immunocompromised mice. 24 hrs later, mice were euthanized and spleens and BM were processed followed by flow cytometry analysis to determine the number of human CD45+ cells that have migrated. Results: We confirmed Siglec-6 overexpression on B cells from CLL patients when compared to B cells from normal donors. Interestingly, we also found expression of sTn on bone marrow stromal cells (BMSCs) derived from CLL patients but not healthy donors. Compared to Siglec-6 + CLL cells, Siglec-6 - CLL cells exhibited significant reduction in adhesion to (~50%) and migration towards (~50%) media containing sTn or sTn + CLL-BMSCs in cell adhesion and trans-well migration assays. Importantly, a Siglec-6 targeted antibody inhibited homing of Siglec-6 + MEC1 cells and primary CLL cells to the spleen and bone marrow in NSG mice (~35%). Mass spectrometry and co-immunoprecipitation analysis in MEC1 cells revealed interaction of Siglec-6 with DOCK8, a guanine nucleotide exchange factor. Stimulation of Siglec-6 + MEC1 cells with sTn resulted in Cdc42 activation and WASP protein recruitment, which are both downstream targets of DOCK8 involved in cell migration. Further, sTn also promoted actin polymerization, an effect that was compromised in Siglec-6 or DOCK8 KO MEC1 cells. Additionally, cell fractionation experiments revealed that Siglec-6 + MEC1 cells had higher levels of DOCK8 at the cell membrane when compared to MEC1 Siglec-6 KO cells, indicating that Siglec-6 may be responsible for tethering DOCK8 to the cell membrane. Conclusions: We have for the first time shown Siglec-6 dependent recruitment of DOCK8 leading to migration and adhesion of B-CLL cells. Siglec-6 signals via DOCK8 to mediate sTn ligand dependent actin polymerization. We have also shown that sTn promotes Cdc42 activation and WASP protein recruitment which are both essential for actin polymerization. Moreover, all these effects were prevented by CRISPR/Cas9 mediated knock out of Siglec-6 or DOCK8 in MEC1 CLL cell line. Thus, Siglec-6 represents a CLL-specific target that opens up new therapeutic avenues to target only malignant B-CLL cells. Ongoing studies are focused on determining molecular mechanisms of Siglec-6 mediated regulation of actin polymerization and CLL-BMSC interactions. [This work was supported by NIH-R21 Grant and Pelotonia Idea grants. JN is a recipient of Pelotonia Graduate Fellowship] Disclosures Byrd: Newave: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria.