ROR1-targeted delivery of miR-29b induces cell cycle arrest and therapeutic benefit in vivo in a CLL mouse model.

Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eµ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.

ROR1-targeted delivery of OSU-2S, a nonimmunosuppressive FTY720 derivative, exerts potent cytotoxicity in mantle-cell lymphoma in vitro and in vivo.

Mantle-cell lymphoma (MCL) remains incurable despite numerous therapeutic advances. OSU-2S, a novel nonimmunosuppressive FTY720 (Fingolimod) derivative, exhibits potent cytotoxicity in MCL cell lines and primary cells. OSU-2S increased the surface expression of CD74, a therapeutic antibody target in MCL cells. OSU-2S, in combination with anti-CD74 antibody milatuzumab, enhanced cytotoxicity in MCL. Moreover, MCL tumor antigen receptor tyrosine kinase-like orphan receptor 1 (ROR1) targeted immunonanoparticle-carrying OSU-2S (2A2-OSU-2S-ILP)-mediated selective cytotoxicity of MCL in vitro, as well as activity in a xenografted mouse model of MCL in vivo. The newly developed OSU-2S delivery using ROR1-directed immunonanoparticles provide selective targeting of OSU-2S to MCL and other ROR1(+) malignancies, sparing normal B cells.

Ibrutinib is an irreversible molecular inhibitor of ITK driving a Th1-selective pressure in T lymphocytes.

Given its critical role in T-cell signaling, interleukin-2-inducible kinase (ITK) is an appealing therapeutic target that can contribute to the pathogenesis of certain infectious, autoimmune, and neoplastic diseases. Ablation of ITK subverts Th2 immunity, thereby potentiating Th1-based immune responses. While small-molecule ITK inhibitors have been identified, none have demonstrated clinical utility. Ibrutinib is a confirmed irreversible inhibitor of Bruton tyrosine kinase (BTK) with outstanding clinical activity and tolerability in B-cell malignancies. Significant homology between BTK and ITK alongside in silico docking studies support ibrutinib as an immunomodulatory inhibitor of both ITK and BTK. Our comprehensive molecular and phenotypic analysis confirms ITK as an irreversible T-cell target of ibrutinib. Using ibrutinib clinical trial samples along with well-characterized neoplastic (chronic lymphocytic leukemia), parasitic infection (Leishmania major), and infectious disease (Listeria monocytogenes) models, we establish ibrutinib as a clinically relevant and physiologically potent ITK inhibitor with broad therapeutic utility. This trial was registered at www.clinicaltrials.gov as #NCT01105247 and #NCT01217749.

Recombination activation gene-2-deficient blastocyst complementation analysis reveals an essential role for nuclear factor I-A transcription factor in T-cell activation.

Nuclear factor I (NFI)-A is a member of the NFI family of transcription factors implicated in regulation of granulocyte differentiation. However, its role in the lymphoid lineage is not known. NFI-A deficiency results in perinatal lethality, thus precluding analysis of the role of NFI-A in lymphocyte development and function. Using recombination activation gene-2-deficient (RAG-2(-/-)) blastocysts and embryonic stem cells with homozygous NFI-A gene deletion, we show an essential role for NFI-A in T-cell activation. NFI-A(-/-)→RAG-2(-/-) chimeric mice had normal distributions of CD4(-)CD8(-) double negative, CD4(+)CD8(+) double positive, CD4(+)CD8(-) and CD4(-)CD8(+)-single positive cells in the thymus and CD4(+)CD8(-) and CD4(-)CD8(+) cells in spleen and lymph nodes. However, NFI-A(-/-)→RAG-2(-)(/)(-) mice had severely reduced thymus size and hypocellularity. The decrease in thymocytes and peripheral T cells in NFI-A(-/-)→RAG-2(-/-) chimeric mice is attributed to proliferative defects associated with decreased blast transformation, CD69 expression and DNA synthesis in response to T antigen receptor stimulation. Interestingly, NFI-A-null T cells showed increased levels of c-myc transcription that is inhibited in response to antigen receptor-mediated activation. These studies demonstrate for the first time a requirement for the NFI-A transcription factor in antigen receptor-induced T-cell activation events.