Preclinical characterization of pirtobrutinib, a highly selective, noncovalent (reversible) BTK inhibitor.

Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell-driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell-driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.

Trisomy 21 consistently activates the interferon response.

Although it is clear that trisomy 21 causes Down syndrome, the molecular events acting downstream of the trisomy remain ill defined. Using complementary genomics analyses, we identified the interferon pathway as the major signaling cascade consistently activated by trisomy 21 in human cells. Transcriptome analysis revealed that trisomy 21 activates the interferon transcriptional response in fibroblast and lymphoblastoid cell lines, as well as circulating monocytes and T cells. Trisomy 21 cells show increased induction of interferon-stimulated genes and decreased expression of ribosomal proteins and translation factors. An shRNA screen determined that the interferon-activated kinases JAK1 and TYK2 suppress proliferation of trisomy 21 fibroblasts, and this defect is rescued by pharmacological JAK inhibition. Therefore, we propose that interferon activation, likely via increased gene dosage of the four interferon receptors encoded on chromosome 21, contributes to many of the clinical impacts of trisomy 21, and that interferon antagonists could have therapeutic benefits.

Schizosaccharomyces pombe histone acetyltransferase Mst1 (KAT5) is an essential protein required for damage response and chromosome segregation.

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature-sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1), and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.

Schizosaccharomyces pombe Rad4/Cut5 protein modification and chromatin binding changes in DNA damage.

The Schizosaccharomyces pombe Rad4/Cut5 protein is essential for DNA replication and checkpoint control. We have analyzed the behavior of the protein during unperturbed DNA replication, in different replication and checkpoint mutant backgrounds and in response to DNA-damaging agents. In an unperturbed cell cycle, Rad4 is chromatin bound and the mobility of the protein is not altered. Rad4 protein level and thus chromatin binding are dependent on a functional DNA polymerase epsilon. In response to replication arrest and DNA damage, the protein is modified in a Rad3-dependent manner. These data indicate that Rad4 undergoes diverse forms of regulation that are distinct in both DNA replication and checkpoint response.

Trypanosoma cruzi Tcp12CKS1 interacts with parasite CRKs and rescues the p13SUC1 fission yeast mutant.

The complex mechanism of cell division in trypanosomatids is not completely fully understood. CRKs (cdc2-related kinases), Cyclins and CKSs (cdc2-kinase subunit) are involved in the progression through the cell cycle. The CKS proteins were first described as components of the cell cycle machinery in yeast and their action has been implicated in the regulation of CDK function. In the present work we identified Tcp12CKS1 a member of the CKS family in the parasite Trypanosoma cruzi. TcCKS1 is expressed in the three forms of T. cruzi. By using anti-Tcp12CKS1 antiserum, protein kinase (PK) activities were immunoprecipitated. The PK activity level varies depending on the stage analyzed, being lower in trypomastigotes and thus suggesting that different stages have different CKS-CRK complexes. Moreover, these PK activities were inhibited by using Flavopiridol, a known CDKs inhibitor. Western blot analyses demonstrated that in the epimastigote stage, p12CKS1 stably interacts with TcCRK1 and TcCRK3. In addition, Tcp12CKS1 was able to rescue the p13SUC1 null mutant of S. pombe. The functional complementation between the CKS proteins of two evolutionary distant organisms supports the role of Tcp12CKS1 as a key regulator in T. cruzi cell cycle.

Schizosaccharomyces pombe mst2+ encodes a MYST family histone acetyltransferase that negatively regulates telomere silencing.

Histone acetylation and deacetylation are associated with transcriptional activity and the formation of constitutively silent heterochromatin. Increasingly, histone acetylation is also implicated in other chromosome transactions, including replication and segregation. We have cloned the only Schizosaccharomyces pombe MYST family histone acetyltransferase genes, mst1(+) and mst2(+). Mst1p, but not Mst2p, is essential for viability. Both proteins are localized to the nucleus and bound to chromatin throughout the cell cycle. Deltamst2 genetically interacts with mutants that affect heterochromatin, cohesion, and telomere structure. Mst2p is a negative regulator of silencing at the telomere but does not affect silencing in the centromere or mating type region. We generated a census of proteins and histone modifications at wild-type telomeres. A histone acetylation gradient at the telomeres is lost in Deltamst2 cells without affecting the distribution of Taz1p, Swi6p, Rad21p, or Sir2p. We propose that the increased telomeric silencing is caused by histone hypoacetylation and/or an increase in the ratio of methylated to acetylated histones. Although telomere length is normal, meiosis is aberrant in Deltamst2 diploid homozygote mutants, suggesting that telomeric histone acetylation contributes to normal meiotic progression.

Suppressors of Bir1p (Survivin) identify roles for the chromosomal passenger protein Pic1p (INCENP) and the replication initiation factor Psf2p in chromosome segregation.

Fission yeast Bir1p/Cut17p/Pbh1p, the homolog of human Survivin, is a conserved chromosomal passenger protein that is required for cell division and cytokinesis. To study how Bir1p promotes accurate segregation of chromosomes, we generated and analyzed a temperature-sensitive allele, bir1-46, and carried out genetic screens to find genes that interact with bir1(+). We identified Psf2p, a component of the GINS complex required for DNA replication initiation, as a high-copy-number suppressor of the bir1-46 growth defect. Loss of Psf2p function by depletion or deletion or by use of a temperature-sensitive allele, psf2-209, resulted in chromosome missegregation that was associated with mislocalization of Bir1p. We also found that the human homolog of Psf2p, PSF2, was required for proper chromosome segregation. In addition, we observed that high-copy-number expression of Pic1p, the fission yeast homolog of INCENP (inner centromere protein), suppressed bir1-46. Pic1p exhibited a localization pattern typical of chromosomal passenger proteins. Deletion of pic1(+) caused chromosome missegregation phenotypes similar to those of bir1-46. Our data suggest that Bir1p and Pic1p act as part of a conserved chromosomal passenger complex and that Psf2p/GINS indirectly affects the localization and function of this complex in chromosome segregation, perhaps through an S-phase role in centromere replication.

Conserved locus-specific silencing functions of Schizosaccharomyces pombe sir2+.

In Schizosaccharomyces pombe, three genes, sir2(+), hst2(+), and hst4(+), encode members of the Sir2 family of conserved NAD(+)-dependent protein deacetylases. The S. pombe sir2(+) gene encodes a nuclear protein that is not essential for viability or for resistance to treatment with UV or a microtubule-destabilizing agent. However, sir2(+) is essential for full transcriptional silencing of centromeres, telomeres, and the cryptic mating-type loci. Chromatin immunoprecipitation results suggest that the Sir2 protein acts directly at these chromosomal regions. Enrichment of Sir2p at silenced regions does not require the HP1 homolog Swi6p; instead, Swi6-GFP localization to telomeres depends in part on Sir2p. The phenotype of sir2 swi6 double mutants supports a model whereby Sir2p functions prior to Swi6p at telomeres and the silent mating-type loci. However, Sir2p does not appear to be essential for the localization of Swi6p to centromeric foci. Cross-complementation experiments showed that the Saccharomyces cerevisiae SIR2 gene can function in place of S. pombe sir2(+), suggesting overlapping deacetylation substrates in both species. These results also suggest that, despite differences in most of the other molecules required, the two distantly related yeast species share a mechanism for targeting Sir2p homologs to silent chromatin.

A screen for Schizosaccharomyces pombe mutants defective in rereplication identifies new alleles of rad4+, cut9+ and psf2+.

Fission yeast mutants defective in DNA replication have widely varying morphological phenotypes. We designed a screen for temperature-sensitive mutants defective in the process of replication regardless of morphology by isolating strains unable to rereplicate their DNA in the absence of cyclin B (Cdc13). Of the 42 rereplication-defective mutants analyzed, we were able to clone complementing plasmids for 10. This screen identified new alleles of the APC subunit cut9(+), the initiation/checkpoint factor rad4(+)/cut5(+), and the first mutant allele of psf2(+), a subunit of the novel GINS replication complex. Other genes identified are likely to play general roles in gene expression and protein localization.

Trypanosoma cruzi poly-zinc finger protein: a novel DNA/RNA-binding CCHC-zinc finger protein.

A poly-zinc finger protein, designated PZFP1 was identified in Trypanosoma cruzi for the first time. The protein has 191 amino acids, contains seven motifs Cys(X)(2)Cys(X)(4)His(X)(4)Cys. A recombinant PZFP1 was generated in E. coli and the expected 21kDa polypeptide co-purified with two other inducible products of about 42 and 63kDa. Western blot analysis of cell extracts using an anti-PZFP1 antibody recognized a major band of 41kDa. Electrophoretic mobility shift analysis demonstrated that both, recombinant and native PZFP1, specifically interact with single-stranded DNA or RNA oligonucleotides carrying recognition sequences of other CCHC proteins. The protein was localized mainly in the cytoplasm and nucleus as observed by indirect immunofluorescence analysis. PZFP1 interacted specifically with a T. cruzi serine-arginine-rich protein (TcSR) in a yeast two-hybrid assay, suggesting a role in pre-mRNA processing.

Evidence for CRK3 participation in the cell division cycle of Trypanosoma cruzi.

Trypanosoma cruzi CRK3 gene encodes a Cdc2p related protein kinase (CRK). To establish if it has a role in the regulation of the parasite cell cycle we studied CRK3 expression and activity throughout three life cycle stages. CRK3 from epimastigote soluble extracts interacted with p13(suc1)-beads. Endogenous CRK3 phosphorylated histone H1 and this activity was inhibited by specific CDK inhibitors: Olomoucine, Flavopiridol and Roscovitine. Flavopiridol partially inhibited the growth of T. cruzi epimastigotes at 50 nM, the lowest concentration used, but even with the highest (5 microM), cell growth was not completely arrested. CRK3 from Flavopiridol-inhibited epimastigote extracts exhibited a dose dependent inhibition of histone H1 phosphorylation. T. cruzi p13(suc1)-binding CRK displayed the same inhibition profile. This suggests that CRK3 is the enzyme responsible for the majority of the kinase activity associated with p13(suc1). CRK3 activity of hydroxyurea (HU) synchronized epimastigotes peaked in G2/M boundary while the kinase activity associated to p13(suc1)-beads increased at the same time point but remained high until late G2/M. In addition, CRK3 expression was constant during the cell cycle. This is a common pattern of CDK activity regulation. Taken together, these results support the idea that CRK3 is involved in control of the cell cycle in T. cruzi.

Different phenotypes in vivo are associated with ATPase motif mutations in Schizosaccharomyces pombe minichromosome maintenance proteins.

The six conserved MCM proteins are essential for normal DNA replication. They share a central core of homology that contains sequences related to DNA-dependent and AAA(+) ATPases. It has been suggested that the MCMs form a replicative helicase because a hexameric subcomplex formed by MCM4, -6, and -7 proteins has in vitro DNA helicase activity. To test whether ATPase and helicase activities are required for MCM protein function in vivo, we mutated conserved residues in the Walker A and Walker B motifs of MCM4, -6, and -7 and determined that equivalent mutations in these three proteins have different in vivo effects in fission yeast. Some mutations reported to abolish the in vitro helicase activity of the mouse MCM4/6/7 subcomplex do not affect the in vivo function of fission yeast MCM complex. Mutations of consensus CDK sites in Mcm4p and Mcm7p also have no phenotypic consequences. Co-immunoprecipitation analyses and in situ chromatin-binding experiments were used to study the ability of the mutant Mcm4ps to associate with the other MCMs, localize to the nucleus, and bind to chromatin. We conclude that the role of ATP binding and hydrolysis is different for different MCM subunits.