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Biomedical subjects

Richard W Kriwacki

Publications and source records attributed to Richard W Kriwacki.

9 recordsLinked to original sources
PubMed2025

KAT6A and KAT7 Histone Acetyltransferase Complexes Are Molecular Dependencies and Therapeutic Targets in NUP98-Rearranged Acute Myeloid Leukemia.

UNLABELLED: NUP98 fusion oncoproteins (FO) are a hallmark of childhood acute myeloid leukemia. NUP98 FOs drive leukemogenesis through phase-separated condensate formation and maintenance of an active chromatin landscape at stem cell-associated genes in cooperation with epigenetic regulators. In this study, we show that MYST family histone acetyltransferase (HAT) complex proteins, including KAT6A/MOZ, KAT7/HBO1, and the common KAT6A/7 complex subunit BRPF1, associate with NUP98 FOs on chromatin and within condensates. MYST HATs are molecular dependencies in NUP98-rearranged (NUP98-r) leukemia, and genetic inactivation or pharmacologic inhibition of KAT6A and KAT7 impairs NUP98-r cell fitness. KAT6A/7 inhibition decreased global H3K23ac levels, displaced NUP98::HOXA9 from chromatin at the Meis1 locus, and led to myeloid cell differentiation. Additionally, KAT6A/7 inhibition decreased leukemic burden in multiple NUP98-r leukemia xenograft mouse models, synergized with menin inhibitor treatment, and was efficacious in menin inhibitor-resistant cells. In summary, we show that MYST family HATs are therapeutically actionable dependencies in NUP98-r acute myeloid leukemia. SIGNIFICANCE: KAT6A and KAT7 associate with NUP98 FOs to drive leukemogenesis. Inhibition of their HAT activity is an effective therapeutic strategy in NUP98-r leukemias, including those resistant to menin inhibition. Moreover, combined KAT6A/7 and menin inhibition is synergistic, supporting clinical translation to improve outcomes for NUP98 FO-driven leukemias.

Humans↗
PubMed2004

p27 binds cyclin-CDK complexes through a sequential mechanism involving binding-induced protein folding.

p27 controls cell proliferation by binding and regulating nuclear cyclin-dependent kinases (CDKs). In addition, p27 interacts with other nuclear and cytoplasmic targets and has diverse biological functions. We seek to understand how the structural and dynamic properties of p27 mediate its several functions. We show that, despite showing disorder before binding its targets, p27 has nascent secondary structure that may have a function in molecular recognition. Binding to Cdk2-cyclin A is accompanied by p27 folding, and kinetic data suggest a sequential mechanism that is initiated by binding to cyclin A. p27 regulates CDK-cyclin complexes involved directly in cell cycle control and does not interact with other closely related CDKs. We show that p27-cyclin interactions are an important determinant of this specificity and propose that the homologous cell cycle regulators p21 and p57 function by a similar sequential, folding-on-binding mechanism.

Amino Acid Sequence↗
PubMed2003

P21Waf1/Cip1 dysfunction in neuroblastoma: a novel mechanism of attenuating G0-G1 cell cycle arrest.

In normal cells in which DNA has been damaged, p53 induces the expression of p21(Waf1/Cip1); p21, in turn, binds to cyclin-dependent kinase 2 (cdk2) and inhibits its function. Inhibition of cdk2 results in cell cycle arrest in G(0)-G(1). Although p53 is transcriptionally active and induces p21 expression in neuroblastoma (NB) cells, the G(0)-G(1) checkpoint is attenuated. Here we report that the mechanism that mediates this defect in NB cells is the inability of p21 to bind to, or inhibit the activity of cdk2. However, when recombinant p21 protein was added to NB cell extracts in vitro, the protein inhibited the activity of cdk2. This finding suggests that endogenous p21 protein in NB cells is inactive and may be bound either to a protein complex or in a conformation that precludes its binding to cdk2. The dysfunction of p21 in NB cells represents a novel mechanism by which the G(0)-G(1) cell cycle checkpoint can be inactivated. This mechanism may be important in regulating the growth of NB and potentially other types of tumors. Cdk inhibitors currently being developed for clinical use may be useful therapy for tumors such as NB in which endogenous cdk inhibitors are defective.

CDC2-CDC28 Kinases↗
PubMed2003

Reversible amyloid formation by the p53 tetramerization domain and a cancer-associated mutant.

The tetramerization domain for wild-type p53 (p53tet-wt) and a p53 mutant, R337H (p53tet-R337H), associated with adrenocortical carcinoma (ACC) in children, can be converted from the soluble native state to amyloid-like fibrils under certain conditions. Circular dichroism, Fourier transform infrared spectroscopy and staining with Congo red and thioflavin T showed that p53tet-wt and p53tet-R337H adopt an alternative beta-sheet conformation (p53tet-wt-beta and p53tet-R337H-beta, respectively), characteristic of amyloid-like fibrils, when incubated at pH 4.0 and elevated temperatures. Electron micrographs showed that the alternative conformations for p53tet-wt (p53tet-wt-beta) and p53tet-R337H (p53tet-R337H-beta) were supramolecular structures best described as "molecular ribbons". FT-IR analysis demonstrated that the mechanism of amyloid-like fibril formation involved unfolding of the p53tet-wt beta-strands, followed by unfolding of the alpha-helices, followed finally by formation of beta-strand-containing structures that other methods showed were amyloid-like ribbons. The mutant, p53tet-R337H, had a significantly higher propensity to form amyloid-like fibrils. Both p53tet-wt (pH 4.0) and p53tet-R337H (pH 4.0 and 5.0), when incubated at room temperature (22 degrees C) for one month, were converted to molecular ribbons. In addition, p53tet-R337H, and not p53tet-wt, readily formed ribbons at pH 4.0 and 37 degrees C over 20 hours. Interestingly, unlike other amyloid-forming proteins, p53tet-wt-beta and p53tet-R337H-beta disassembled and refolded to the native tetramer conformation when the solution pH was raised from 4.0 to 8.5. Although fibril formation at pH 4.0 was concentration and temperature-dependent, fibril disassembly at pH 8.5 was independent of both. Finally, we propose that the significantly higher propensity of the mutant to form ribbons, compared to the wild-type, may provide a possible mechanism for the observed nuclear accumulation of p53 in ACC cells and other cancerous cells.

Amyloid↗
PubMed2003

Peptides derived from two dynamically disordered proteins self-assemble into amyloid-like fibrils.

Short peptides derived from p14ARF and Hdm2 (14 and 15 amino acids in length, respectively), two cancer associated proteins, have been found to co-assemble into amyloid-like structures. Larger protein domains containing these peptide segments interact in cells and also undergo a disorder-to-order transition upon binding in vitro. In contrast to the association of beta-strand assemblies with amyloid diseases, the system described herein utilizes the formation of binary, extended beta-strands as a novel mechanism of biomolecular assembly. The beta-strand-containing fibrils formed from these peptides may allow the directed assembly of decorated fibrils with applications as biological nanostructures.

Amino Acid Sequence↗
PubMed2003

Structure, activity, and distribution of fish osteocalcin.

Osteocalcin (bone Gla protein) is an extracellular matrix protein synthesized by osteoblasts that is a marker of bone. Osteocalcin probably originated in the ancestors of Teleostei or bony fish and of the Tetrapoda or amphibians, reptiles, birds, and mammals. We have characterized the Cyprinus carpio (carp) osteocalcin for mineral binding to hydroxyapatite, amino acid sequence, and extent of secondary structure. Hydroxyapatite binding is enhanced in the presence of calcium. The alpha-helical content of teleost osteocalcin increases and beta-sheet structure decreases upon calcium binding, similar to findings in calf osteocalcin. The gene structure and primary sequence of prepro-osteocalcin from 2 pufferfish compared with carp shows that there are many conserved features in teleost osteocalcin genes. Using an immunoassay for carp osteocalcin, we determined that the relative content of osteocalcin is highest in dorsal fin spines and other bones and lowest in scales. The carp osteocalcin antibodies, cross-reactive to other species of fish, were used to study the role of osteocalcin in teleost model systems.

Amino Acid Sequence↗
PubMed2002

Structure and dynamics of thioguanine-modified duplex DNA.

Mercaptopurine and thioguanine, two of the most widely used antileukemic agents, exert their cytotoxic, therapeutic effects by being incorporated into DNA as deoxy-6-thioguanosine. However, the molecular mechanism(s) by which incorporation of these thiopurines into DNA translates into cytotoxicity is unknown. The solution structure of thioguanine-modified duplex DNA presented here shows that the effects of the modification on DNA structure were subtle and localized to the modified base pair. Specifically, thioguanine existed in the keto form, formed weakened Watson-Crick hydrogen bonds with cytosine and caused a modest approximately 10 degrees opening of the modified base pair toward the major groove. In contrast, thioguanine significantly altered base pair dynamics, causing an approximately 80-fold decrease in the base pair lifetime with cytosine compared with normal guanine. This perturbation was consistent with the approximately 6 degrees C decrease in DNA melting temperature of the modified oligonucleotide, the 1.13 ppm upfield shift of the thioguanine imino proton resonance, and the large increase in the exchange rate of the thioguanine imino proton with water. Our studies provide new mechanistic insight into the effects of thioguanine incorporation into DNA at the level of DNA structure and dynamics, provide explanations for the effects of thioguanine incorporation on the activity of DNA-processing enzymes, and provide a molecular basis for the specific recognition of thioguanine-substituted sites by proteins. These combined effects likely cooperate to produce the cellular responses that underlie the therapeutic effects of thiopurines.

Antimetabolites, Antineoplastic↗
PubMed2002

A novel mechanism of tumorigenesis involving pH-dependent destabilization of a mutant p53 tetramer.

The p53 tumor suppressor requires tetramerization to function as an initiator of cell cycle arrest and/or apoptosis. Children in southern Brazil that exhibit an elevated incidence of adrenocortical carcinoma (ACC) harbor an Arg 337 to His mutation within the tetramerization domain of p53 (p53-R337H; 35 of 36 patients). The mutant tetramerization domain (p53tet-R337H) adopts a native-like fold but is less stable than the wild type domain (p53tet-wt). Furthermore, the stability of p53tet-R337H is highly sensitive to pH in the physiological range; this sensitivity correlates with the protonation state of the mutated His 337. These results demonstrate a pH-sensitive molecular defect of p53 (R337H), suggesting that pH-dependent p53 dysfunction is the molecular basis for these cases of ACC in Brazilian children.

Adrenocortical Carcinoma↗