Modulation of RNA splicing associated with Wnt signaling pathway using FD-895 and pladienolide B.

Alterations in RNA splicing are associated with different malignancies, including leukemia, lymphoma, and solid tumors. The RNA splicing modulators such as FD-895 and pladienolide B have been investigated in different malignancies to target/modulate spliceosome for therapeutic purpose. Different cell lines were screened using an RNA splicing modulator to test in vitro cytotoxicity and the ability to modulate RNA splicing capability via induction of intron retention (using RT-PCR and qPCR). The Cignal Finder Reporter Array evaluated [pathways affected by the splice modulators in HeLa cells. Further, the candidates associated with the pathways were validated at protein level using western blot assay, and gene-gene interaction studies were carried out using GeneMANIA. We show that FD-895 and pladienolide B induces higher apoptosis levels than conventional chemotherapy in different solid tumors. In addition, both agents modulate Wnt signaling pathways and mRNA splicing. Specifically, FD-895 and pladienolide B significantly downregulates Wnt signaling pathway-associated transcripts (GSK3ß and LRP5) and both transcript and proteins including LEF1, CCND1, LRP6, and pLRP6 at the transcript, total protein, and protein phosphorylation's levels. These results indicate FD-895 and pladienolide B inhibit Wnt signaling by decreasing LRP6 phosphorylation and modulating mRNA splicing through induction of intron retention in solid tumors.

RNA Splicing Modulation Selectively Impairs Leukemia Stem Cell Maintenance in Secondary Human AML.

Age-related human hematopoietic stem cell (HSC) exhaustion and myeloid-lineage skewing promote oncogenic transformation of hematopoietic progenitor cells into therapy-resistant leukemia stem cells (LSCs) in secondary acute myeloid leukemia (AML). While acquisition of clonal DNA mutations has been linked to increased rates of secondary AML for individuals older than 60 years, the contribution of RNA processing alterations to human hematopoietic stem and progenitor aging and LSC generation remains unclear. Comprehensive RNA sequencing and splice-isoform-specific PCR uncovered characteristic RNA splice isoform expression patterns that distinguished normal young and aged human stem and progenitor cells (HSPCs) from malignant myelodysplastic syndrome (MDS) and AML progenitors. In splicing reporter assays and pre-clinical patient-derived AML models, treatment with a pharmacologic splicing modulator, 17S-FD-895, reversed pro-survival splice isoform switching and significantly impaired LSC maintenance. Therapeutic splicing modulation, together with monitoring splice isoform biomarkers of healthy HSPC aging versus LSC generation, may be employed safely and effectively to prevent relapse, the leading cause of leukemia-related mortality.

Selectivity in Small Molecule Splicing Modulation.

The dysregulation of RNA splicing is a molecular hallmark of disease, including different and often complex cancers. While gaining recognition as a target for therapeutic discovery, understanding the complex mechanisms guiding RNA splicing remains a challenge for chemical biology. The discovery of small molecule splicing modulators has recently enabled an evaluation of the mechanisms of aberrant splicing. We now report on three unique features within the selectivity of splicing modulators. First, we provide evidence that structural modifications within a splicing modulator can alter the splicing of introns in specific genes differently. These studies indicate that structure activity relationships not only have an effect on splicing activity but also include specificity for specific introns within different genes. Second, we find that these splicing modulators also target the mRNAs encoding components of the spliceosome itself. Remarkably, this effect includes the genes for the SF3B complex, a target of pladienolide B and related splicing modulators. Finally, we report on the first observation of a temporal phenomenon associated with small molecule splicing modulation. Combined, these three observations provide an important new perspective for the exploration of splicing modulation in terms of both future medicinal chemistry programs as well as understanding the key facets underlying its timing.

Targeting the spliceosome in chronic lymphocytic leukemia with the macrolides FD-895 and pladienolide-B.

RNA splicing plays a fundamental role in human biology. Its relevance in cancer is rapidly emerging as demonstrated by spliceosome mutations that determine the prognosis of patients with hematologic malignancies. We report studies using FD-895 and pladienolide-B in primary leukemia cells derived from patients with chronic lymphocytic leukemia and leukemia-lymphoma cell lines. We found that FD-895 and pladienolide-B induce an early pattern of mRNA intron retention - spliceosome modulation. This process was associated with apoptosis preferentially in cancer cells as compared to normal lymphocytes. The pro-apoptotic activity of these compounds was observed regardless of poor prognostic factors such as Del(17p), TP53 or SF3B1 mutations and was able to overcome the protective effect of culture conditions that resemble the tumor microenvironment. In addition, the activity of these compounds was observed not only in vitro but also in vivo using the A20 lymphoma murine model. Overall, these findings give evidence for the first time that spliceosome modulation is a valid target in chronic lymphocytic leukemia and provide an additional rationale for the development of spliceosome modulators for cancer therapy.

Stabilized cyclopropane analogs of the splicing inhibitor FD-895.

Targeting the spliceosome with small molecule inhibitors provides a new avenue to target cancer by intercepting alternate splicing pathways. Although our understanding of alternate mRNA splicing remains poorly understood, it provides an escape pathway for many cancers resistant to current therapeutics. These findings have encouraged recent academic and industrial efforts to develop natural product spliceosome inhibitors, including FD-895 (1a), pladienolide B (1b), and pladienolide D (1c), into next-generation anticancer drugs. The present study describes the application of semisynthesis and total synthesis to reveal key structure-activity relationships for the spliceosome inhibition by 1a. This information is applied to deliver new analogs with improved stability and potent activity at inhibiting splicing in patient derived cell lines.

Metabolic perturbation of an essential pathway: evaluation of a glycine precursor of coenzyme A.

Pantetheine and its corresponding disulfide pantethine play a key role in metabolism as building blocks of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and nonribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we show that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655ΔpanC lacking a functional pantothenate synthetase. Using mass spectrometry, we show that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogues.

Structure of FD-895 revealed through total synthesis.

The total synthesis of FD-895 was completed through a strategy that featured the use of a tandem esterification ring-closing metathesis (RCM) process to construct the 12-membered macrolide and a modified Stille coupling to append the side chain. These studies combined with detailed analysis of all four possible C16-C17 stereoisomers were used to confirm the structure of FD-895 and identify an analog with an enhanced subnanomolar bioactivity.

Total synthesis of (±)-hirsutine: application of phosphine-catalyzed imine-allene [4 + 2] annulation.

The total synthesis of the indole alkaloid hirsutine has been achieved, with a key step being the application of our phosphine-catalyzed [4 + 2] annulation of an imine with ethyl α-methylallenoate. From commercially available indole-2-carboxaldehyde, the target was synthesized in 14 steps and 6.7% overall yield.