Phase 1 study of JNJ-64619178, a protein arginine methyltransferase 5 inhibitor, in patients with lower-risk myelodysplastic syndromes.

Splicing factor (SF) gene mutations are frequent in myelodysplastic syndromes (MDS), and agents that modulate RNA splicing are hypothesized to provide clinical benefit. JNJ-64619178, a protein arginine methyltransferase 5 (PRMT5) inhibitor, was evaluated in patients with lower-risk (LR) MDS in a multi-part, Phase 1, multicenter study. The objectives were to determine a tolerable dose and to characterize safety, pharmacokinetics, pharmacodynamics, and preliminary clinical activity.

Phase 1 Study of JNJ-64619178, a Protein Arginine Methyltransferase 5 Inhibitor, in Advanced Solid Tumors.

Purpose: In this first-in-human, Phase 1, open-label, multicenter study, we evaluated JNJ-64619178, a selective and potent PRMT5 inhibitor, in patients with advanced malignant solid tumors or non-Hodgkin lymphomas (NHL). The primary objective was to evaluate the safety and to identify a recommended Phase 2 dose (RP2D) of JNJ-64619178.

Patients And Methods: Adult patients with treatment-refractory advanced solid tumors or NHL and measurable disease received escalating doses of JNJ-64619178 following two schedules (Schedule A: 14 days on/7 days off; Schedule B: every day on a 21-day cycle).

High-affinity FRβ-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FRβ)-specific single-chain variable fragment (2.

Eltrombopag modulates reactive oxygen species and decreases acute myeloid leukemia cell survival.

Previous studies have demonstrated that the small molecule thrombopoietin (TPO) mimetic, eltrombopag (E), induces apoptosis in acute myeloid leukemia (AML) cells. Here, we sought to define the mechanism of the anti-leukemic effect of eltrombopag. Our studies demonstrate that, at a concentration of 5 μM E in 2% serum, E induces apoptosis in leukemia cells by triggering PARP cleavage and activation of caspase cascades within 2-6 hours.

Targeting of folate receptor β on acute myeloid leukemia blasts with chimeric antigen receptor-expressing T cells.

T cells expressing a chimeric antigen receptor (CAR) can produce dramatic results in lymphocytic leukemia patients; however, therapeutic strategies for myeloid leukemia remain limited. Folate receptor β (FRβ) is a myeloid-lineage antigen expressed on 70% of acute myeloid leukemia (AML) patient samples. Here, we describe the development and evaluation of the first CARs specific for human FRβ (m909) in vitro and in vivo.

Intrinsic resistance to JAK2 inhibition in myelofibrosis.

Purpose: Recent results have shown that myeloproliferative neoplasms (MPN) are strongly associated with constitutive activation of the Janus-activated kinase (JAK)2 tyrosine kinase. However, JAK2 inhibitors currently approved or under development for treating myeloproliferative neoplasms do not selectively deplete the malignant clone, and the inhibition of activity of the drug target (JAK2) has not been rigorously evaluated in the clinical studies. Therefore, in this study we developed an in vitro assay to gain insight into how effectively JAK2 activity is inhibited in the samples of patients.

Short hairpin RNA screen reveals bromodomain proteins as novel targets in acute myeloid leukemia.

Targeting chromatin regulators for the treatment of malignancies has shown great promise, but also revealed significant challenges. By employing an elegant shRNA screen and a selective pharmacological inhibitor, a recent study published in Nature establishes the bromodomain protein Brd4 as novel target in acute myeloid leukemia (AML).

Phenylpyrrolocytosine as an unobtrusive base modification for monitoring activity and cellular trafficking of siRNA.

6-Phenylpyrrolocytosine (PhpC) is a cytosine mimic with excellent base-pairing fidelity, thermal stability, and high fluorescence. In this work, PhpC-containing small interfering RNAs (siRNAs) are shown to possess thermal stability and gene silencing activity that is virtually identical to that of natural siRNA. The emissive properties of PhpC allow the cellular trafficking of PhpC-containing siRNAs to be monitored by fluorescence microscopy.

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing.

We report that combining a DNA analog (2'F-ANA) with rigid RNA analogs [2'F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1-1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides.

c-Myb binds MLL through menin in human leukemia cells and is an important driver of MLL-associated leukemogenesis.

Mixed-lineage leukemia (MLL) is a proto-oncogene frequently involved in chromosomal translocations associated with acute leukemia. These chromosomal translocations commonly result in MLL fusion proteins that dysregulate transcription. Recent data suggest that the MYB proto-oncogene, which is an important regulator of hematopoietic cell development, has a role in leukemogenesis driven by the MLL-ENL fusion protein, but exactly how is unclear.

A prototype nonpeptidyl, hydrazone class, thrombopoietin receptor agonist, SB-559457, is toxic to primary human myeloid leukemia cells.

Biologic characterization of SB-559457 (SB), a nonpeptidyl hydrazone class of thrombopoietin receptor (Mpl) agonist, revealed toxicity toward human leukemia cells. Antiproliferative effects followed by significant, nonapoptotic, cell death within 72 hours occurred in 24 of 26 acute myeloid leukemia, 0 of 6 acute lymphoblastic leukemia, and 3 of 6 chronic myeloid leukemia patient samples exposed to SB, but not recombinant human thrombopoietin (rhTpo), in liquid suspension culture. Further investigation revealed increased phosphorylation of p70S6/S6 kinases in SB-, but not in rhTpo-, treated cells.

The c-myb proto-oncogene and microRNA-15a comprise an active autoregulatory feedback loop in human hematopoietic cells.

The c-myb proto-oncogene encodes an obligate hematopoietic cell transcription factor important for lineage commitment, proliferation, and differentiation. Given its critical functions, c-Myb regulatory factors are of great interest but remain incompletely defined. Herein we show that c-Myb expression is subject to posttranscriptional regulation by microRNA (miRNA)-15a.

Trisomy 21 enhances human fetal erythro-megakaryocytic development.

Children with Down syndrome exhibit 2 related hematopoietic diseases: transient myeloproliferative disorder (TMD) and acute megakaryoblastic leukemia (AMKL). Both exhibit clonal expansion of blasts with biphenotypic erythroid and megakaryocytic features and contain somatic GATA1 mutations. While altered GATA1 inhibits erythro-megakaryocytic development, less is known about how trisomy 21 impacts blood formation, particularly in the human fetus where TMD and AMKL originate.

Possible mechanisms of improved radiation response by cytotoxic RNase, Onconase, on A549 human lung cancer xenografts of nude mice.

The cytotoxic RNase, Onconase (ONC), isolated from amphibian oocytes, was used to study its effect on the radiation response in A549 human NSCLC in vitro and in vivo. In cell culture studies, we found that ONC increased the radiation response by ONC-induced inhibition of O2 consumption (QO2). The occurrence of apoptosis was increased by ONC and was dependent on dosages and time exposure (measured by a Tunnel in situ cell death detection assay).

The C-MYB locus is involved in chromosomal translocation and genomic duplications in human T-cell acute leukemia (T-ALL), the translocation defining a new T-ALL subtype in very young children.

The C-Myb transcription factor is essential for hematopoiesis, including in the T-cell lineage. The C-Myb locus is a common site of retroviral insertional mutagenesis, however no recurrent genomic involvement has been reported in human malignancies. Here, we identified 2 types of genomic alterations involving the C-MYB locus at 6q23 in human T-cell acute leukemia (T-ALL).

Antitumor efficacy of the cytotoxic RNase, ranpirnase, on A549 human lung cancer xenografts of nude mice.

Background: The cytotoxic RNase, ranpirnase (ONCONASE, ONC), may have promising therapeutic implication as an alternative for cisplatin for the treatment of lung cancer, due to inhibition of protein synthesis by t-RNA cleavage.

Materials And Methods: A549 and NCI-H1975 human NSCLC cell lines were cultured in the presence and absence of ONC. Cytotoxicity was monitored using a clonogenic assay.

Design of antisense oligonucleotides and short interfering RNA duplexes (siRNA) targeted to BCL6 mRNA: towards rational drug development for specific lymphoma subsets.

Many algorithms based on computational analysis and thermodynamic parameters have been developed to predict the secondary structure of RNA. Still, many antisense oligodeoxynucleotides (AS ODNs), or siRNA molecules designed according to these predictions fail to silence the intended target, whereas other, not fulfilling those criteria prove highly active. We have developed a reliable mapping strategy, which allows us to predict the sites accessible for hybridization within target mRNA in vitro and in vivo.

c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression.

Myb family proteins are ubiquitously expressed transcription factors. In mammalian cells, they play a critical role in regulating the G(1)/S cell cycle transition but their role in regulating other cell cycle checkpoints is incompletely defined. Herein, we report experiments which demonstrate that c-Myb upregulates cyclin B1 expression in normal and malignant human hematopoietic cells.

Nucleic acid therapeutics for hematologic malignancies--theoretical considerations.

Our work is motivated by the belief that RNA targeted gene silencing agents can be developed into effective drugs for treating hematologic malignancies. In many experimental systems, antisense nucleic acids of various composition, including antisense oligodeoxynucleotides (AS ODNs) and short interfering RNA (siRNA), have been shown to perturb gene expression in a sequence specific manner. Nevertheless, our clinical experience, and those of others, have led us to conclude that the antisense nucleic acids (ASNAs) we, and others, employ need to be optimized with regard to intracellular delivery, targeting, chemical composition, and efficiency of mRNA destruction.

Progress in the development of nucleic acid therapeutics.

Abnormal gene expression is a hallmark of many diseases. Gene-specific downregulation of aberrant genes could be useful therapeutically and potentially less toxic than conventional therapies due its specificity. Over the years, many strategies have been proposed for silencing gene expression in a gene-specific manner.

2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing.

To be effective in vivo, antisense oligonucleotides (AS ON) should be nuclease resistant, form stable ON/RNA duplexes and support ribonuclease H mediated heteroduplex cleavage, all with negligible non-specific effects on cell function. We report herein that AS ONs containing a 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) sugar modification not only meet these criteria, but have the added advantage of maintaining high intracellular concentrations for prolonged periods of time which appears to promote longer term gene silencing. To demonstrate this, we targeted the c-MYB protooncogene's mRNA in human leukemia cells with fully phosphorothioated 2'F-ANA-DNA chimeras (PS-2'FANA-DNA) and compared their gene silencing efficiency with AS ON containing unmodified nucleosides (PS-DNA).

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact.

Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules.

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression.

Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells.

We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80-95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable.