A Multicenter, Open-Label, Phase I/II Study of FN-1501 in Patients with Advanced Solid Tumors.

BACKGROUND: FN-1501, a potent inhibitor of receptor FMS-like tyrosine kinase 3 (FLT3) and CDK4/6, KIT, PDGFR, VEGFR2, ALK, and RET tyrosine kinase proteins, has demonstrated significant in vivo activity in various solid tumor and leukemia human xenograft models. Anomalies in FLT3 have an established role as a therapeutic target where the gene has been shown to play a critical role in the growth, differentiation, and survival of various cell types in hematopoietic cancer and have shown promise in various solid tumors. An open-label, Phase I/II study (NCT03690154) was designed to evaluate the safety and PK profile of FN-1501 as monotherapy in patients (pts) with advanced solid tumors and relapsed, refractory (R/R) AML. METHODS: Pts received FN-1501 IV three times a week for 2 weeks, followed by 1 week off treatment in continuous 21-day cycles. Dose escalation followed a standard 3 + 3 design. Primary objectives include the determination of the maximum tolerated dose (MTD), safety, and recommended Phase 2 dose (RP2D). Secondary objectives include pharmacokinetics (PK) and preliminary anti-tumor activity. Exploratory objectives include the relationship between pharmacogenetic mutations (e.g., FLT3, TP53, KRAS, NRAS, etc.), safety, and efficacy; as well as an evaluation of the pharmacodynamic effects of treatment with FN-1501. Dose expansion at RP2D further explored the safety and efficacy of FN-1501 in this treatment setting. RESULTS: A total of 48 adult pts with advanced solid tumors (N = 47) and AML (N = 1) were enrolled at doses ranging from 2.5 to 226 mg IV three times a week for two weeks in 21-day cycles (2 weeks on and 1 week off treatment). The median age was 65 years (range 30-92); 57% were female and 43% were male. The median number of prior lines of treatment was 5 (range 1-12). Forty patients evaluable for dose-limiting toxicity (DLT) assessment had a median exposure of 9.5 cycles (range 1-18 cycles). Treatment-related adverse events (TRAEs) were reported for 64% of the pts. The most common treatment-emergent adverse events (TEAEs), defined as those occurring in ≥20% of pts, primarily consisted of reversible Grade 1-2 fatigue (34%), nausea (32%), and diarrhea (26%). The most common Grade ≥3 events occurring in ≥5% of pts consisted of diarrhea and hyponatremia. Dose escalation was discontinued due to DLTs of Grade 3 thrombocytopenia (N = 1) and Grade 3 infusion-related reaction (N = 1) occurring in 2 pts. The maximum tolerated dose (MTD) was determined to be 170 mg. CONCLUSIONS: FN-1501 demonstrated reasonable safety, tolerability, and preliminary activity against solid tumors in doses up to 170 mg. Dose escalation was terminated based on 2 DLTs occurring at the 226 mg dose level.

A Phase I Dose-Escalation and Dose-Expansion Study of FCN-437c, a Novel CDK4/6 Inhibitor, in Patients with Advanced Solid Tumors.

A phase I study evaluated the safety, tolerability, and maximum-tolerated dose (MTD)/recommended phase II dose (RP2D) of FCN-437c, a novel, orally available cyclin-dependent kinase inhibitor (CDK4/6i), in participants with advanced/metastatic solid tumors (aSTs). FCN-437c was escalated from 50 mg (once daily [QD] on days 1-21 of 28-day cycles) to the MTD/RP2D. In the dose-expansion phase, patients with CDK4/6i-treated breast cancer, or KRAS-mutant (KRASmut) non-small-cell lung cancer (NSCLC) received the MTD. Twenty-two patients were enrolled. The most common tumors in the dose-escalation phase (n = 15) were breast, colorectal, and lung (each n = 4 [27.3%]). The dose-expansion phase included five (71.4%) patients with breast cancer and two (28.6%) with KRASmut NSCLC. Twenty (90.9%) participants experienced FCN-437c-related adverse events. Dose-limiting toxicities occurred in two (33.3%) participants (200-mg dose, dose-escalation phase): grade 3 neutropenia and grade 4 neutrophil count decreased. Due to toxicities reported at 150 mg QD, the MTD was de-escalated to 100 mg QD. One (4.5%) participant (KRASmut NSCLC, 100-mg dose) achieved a partial response lasting 724+ days, and five (22.7%) had stable disease lasting 56+ days. In conclusion, FCN-437c was well tolerated with encouraging signs of antitumor activity and disease control. Further exploration of FCN-437c in aSTs is warranted.

Distinct factors control histone variant H3.3 localization at specific genomic regions.

The incorporation of histone H3 variants has been implicated in the epigenetic memory of cellular state. Using genome editing with zinc-finger nucleases to tag endogenous H3.3, we report genome-wide profiles of H3 variants in mammalian embryonic stem cells and neuronal precursor cells. Genome-wide patterns of H3.3 are dependent on amino acid sequence and change with cellular differentiation at developmentally regulated loci. The H3.3 chaperone Hira is required for H3.3 enrichment at active and repressed genes. Strikingly, Hira is not essential for localization of H3.3 at telomeres and many transcription factor binding sites. Immunoaffinity purification and mass spectrometry reveal that the proteins Atrx and Daxx associate with H3.3 in a Hira-independent manner. Atrx is required for Hira-independent localization of H3.3 at telomeres and for the repression of telomeric RNA. Our data demonstrate that multiple and distinct factors are responsible for H3.3 localization at specific genomic locations in mammalian cells.

Interferon-alpha induces the growth inhibition of human T-cell leukaemia line Jurkat through p38alpha and p38beta.

Interferon alpha (IFN-alpha) modulates the proliferation of different human tumour cell lines. It has been shown that IFN-alpha induces the growth inhibition of T-cell acute lymphoblastic leukaemia (T-ALL). However, its intracellular signalling mechanisms remain unknown. This study found that IFN-alpha inhibited the cell proliferation of human T-ALL cell line Jurkat in a dose- and time-dependent manner. A p38 inhibitor (SB203580), but not an extracellular signal-regulated kinase 1/2 inhibitor (PD98059) or c-Jun N-terminal kinase inhibitor (SP600125), eliminated IFN-alpha inhibition of Jurkat cell proliferation, indicating that p38 pathway is crucial for IFN-alpha-mediated growth inhibition. SB203580 targeted two p38 isoforms, p38alpha and p38beta. The expression of p38alpha and p38beta mRNA in Jurkat cells was examined by reverse transcriptase-polymerase chain reaction. The kinase activity of p38alpha and p38beta was activated by IFN-alpha in Jurkat cells. To investigate the role of p38alpha and p38beta isoforms in IFN-alpha-mediated growth inhibition, we generated stable clones that overexpressed the dominant-negative p38 isoform, p38alpha(AF) or p38beta(AF), in Jurkat cells. Overexpression of p38alpha(AF) or p38beta(AF) inhibited IFN-alpha-mediated p38 kinase activity and growth inhibition in Jurkat cells. Similarly, down-regulation of either p38alpha or p38beta by isoform-specific small interference RNAs also reduced IFN-alpha-mediated growth inhibition. These results demonstrate that IFN-alpha can regulate growth inhibition of Jurkat cells through p38alpha and p38beta.

Targeted transgene integration in plant cells using designed zinc finger nucleases.

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.

Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases.

Homozygosity for the naturally occurring Delta32 deletion in the HIV co-receptor CCR5 confers resistance to HIV-1 infection. We generated an HIV-resistant genotype de novo using engineered zinc-finger nucleases (ZFNs) to disrupt endogenous CCR5. Transient expression of CCR5 ZFNs permanently and specifically disrupted approximately 50% of CCR5 alleles in a pool of primary human CD4(+) T cells. Genetic disruption of CCR5 provided robust, stable and heritable protection against HIV-1 infection in vitro and in vivo in a NOG model of HIV infection. HIV-1-infected mice engrafted with ZFN-modified CD4(+) T cells had lower viral loads and higher CD4(+) T-cell counts than mice engrafted with wild-type CD4(+) T cells, consistent with the potential to reconstitute immune function in individuals with HIV/AIDS by maintenance of an HIV-resistant CD4(+) T-cell population. Thus adoptive transfer of ex vivo expanded CCR5 ZFN-modified autologous CD4(+) T cells in HIV patients is an attractive approach for the treatment of HIV-1 infection.

Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery.

Achieving the full potential of zinc-finger nucleases (ZFNs) for genome engineering in human cells requires their efficient delivery to the relevant cell types. Here we exploited the infectivity of integrase-defective lentiviral vectors (IDLV) to express ZFNs and provide the template DNA for gene correction in different cell types. IDLV-mediated delivery supported high rates (13-39%) of editing at the IL-2 receptor common gamma-chain gene (IL2RG) across different cell types. IDLVs also mediated site-specific gene addition by a process that required ZFN cleavage and homologous template DNA, thus establishing a platform that can target the insertion of transgenes into a predetermined genomic site. Using IDLV delivery and ZFNs targeting distinct loci, we observed high levels of gene addition (up to 50%) in a panel of human cell lines, as well as human embryonic stem cells (5%), allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.

An improved zinc-finger nuclease architecture for highly specific genome editing.

Genome editing driven by zinc-finger nucleases (ZFNs) yields high gene-modification efficiencies (>10%) by introducing a recombinogenic double-strand break into the targeted gene. The cleavage event is induced using two custom-designed ZFNs that heterodimerize upon binding DNA to form a catalytically active nuclease complex. Using the current ZFN architecture, however, cleavage-competent homodimers may also form that can limit safety or efficacy via off-target cleavage. Here we develop an improved ZFN architecture that eliminates this problem. Using structure-based design, we engineer two variant ZFNs that efficiently cleave DNA only when paired as a heterodimer. These ZFNs modify a native endogenous locus as efficiently as the parental architecture, but with a >40-fold reduction in homodimer function and much lower levels of genome-wide cleavage. This architecture provides a general means for improving the specificity of ZFNs as gene modification reagents.

Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases.

Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.

JAK1 N-terminus binds to conserved Box 1 and Box 2 motifs of cytokine receptor common beta subunit but signal activation requires JAK1 C-terminus.

The human interleukin-3 receptor (hIL-3R) consists of a unique alpha subunit (hIL-3Ralpha) and a common beta subunit (betac). Binding of IL-3 to IL-3R activates Janus kinases JAK1 and JAK2. Our previously study showed that JAK2 and JAK1 were constitutively associated with the hIL-3Ralpha and betac subunits, respectively. In this study, we further demonstrate that JAK2 binds to the intracellular domain of hIL-3Ralpha and JAK1 binds to the Box 1 and Box 2 motifs of betac using GST-hIL-3R fusion proteins in pull-down assays. JAK1 mutational analysis revealed that its JH7-3 domains bound directly to the Box 1 and Box 2 motifs of betac. We further examined the role of JAK1 JH7-3 domains in JAK1 and JAK2-mediated signaling using the CDJAKs fusion proteins, which consisted of a CD16 extracellular domain, a CD7 transmembrane domain, and either JAK1 (CDJAK1), JAK2 (CDJAK2), or JAK1-JH7-3 domains (CDJAK1-JH7-3) as intracellular domains. Anti-CD16 antibody crosslinking of wild type fusion proteins CDJAK1 with CDJAK2 could mimic IL-3 signaling, however, the crosslinking of fusion proteins CDJAK1-JH7-3 with CDJAK2 failed to activate downstream proteins. These results suggest that the JAK1-JH7-3 domains are required for betac interaction and abolish wild type JAK1 and JAK2-mediated signaling.

Highly efficient endogenous human gene correction using designed zinc-finger nucleases.

Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rgamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.

Zinc-finger protein-targeted gene regulation: genomewide single-gene specificity.

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics.