bims-numges 2022-01-23 papers

bims-numges

Biomed News

on Nucleotide metabolism and genome stability

Issue of 2022‒01‒23
thirty-one papers selected by
Sean Rudd
Karolinska Institutet

WEE1 kinase protects the stability of stalled DNA replication forks by limiting CDK2 activity.
XRCC1 Prevents Replication Fork Instability during Misincorporation of the DNA Demethylation Bases 5-Hydroxymethyl-2'-Deoxycytidine and 5-Hydroxymethyl-2'-Deoxyuridine.
CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal.
Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ.
3D Genome Organization: Causes and Consequences for DNA Damage and Repair.
Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex.
Structural Insights into the Specificity of 8-Oxo-7,8-dihydro-2'-deoxyguanosine Bypass by Family X DNA Polymerases.
Transcription-Replication Coordination.
Recent advances in DDR (DNA damage response) inhibitors for cancer therapy.
CDK5RAP3, a New BRCA2 Partner That Regulates DNA Repair, Is Associated with Breast Cancer Survival.
Disrupting PHF8-TOPBP1 connection elicits a breast tumor-specific vulnerability to chemotherapeutics.
53BP1-ACLY-SLBP-coordinated activation of replication-dependent histone biogenesis maintains genomic integrity.
Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble.
CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism.
TOPORS-mediated RAD51 SUMOylation facilitates homologous recombination repair.
53BP1 regulates heterochromatin through liquid phase separation.
Interactions with stromal cells promote a more oxidized cancer cell redox state in pancreatic tumors.
Targeting squalene epoxidase interrupts homologous recombination via the ER stress response and promotes radiotherapy efficacy.
Pre-clinical activity of the oral DNA-PK inhibitor, peposertib (M3814), combined with radiation in xenograft models of cervical cancer.
POLIE suppresses telomerase-mediated telomere G-strand extension and helps ensure proper telomere C-strand synthesis in trypanosomes.
The fork protection complex recruits FACT to reorganize nucleosomes during replication.
HMCES safeguards genome integrity and long-term self-renewal of hematopoietic stem cells during stress responses.
Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.
Therapeutic implications of germline vulnerabilities in DNA repair for precision oncology.
SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia.
TRIM21 suppresses CHK1 activation by preferentially targeting CLASPIN for K63-linked ubiquitination.
Validation of a high throughput screening assay to identify small molecules that target the eukaryotic replicative helicase.
BI-2536 Promotes Neuroblastoma Cell Death via Minichromosome Maintenance Complex Components 2 and 10.
Targeting glutamine metabolism network for the treatment of therapy-resistant prostate cancer.
Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast.
Treatment Outcomes with Purine Nucleoside Analog Alone or with Rituximab for Hairy Cell Leukemia at First Relapse.

Cell Rep. 2022 Jan 18. pii: S2211-1247(21)01773-3. [Epub ahead of print]38(3): 110261

WEE1 kinase protects the stability of stalled DNA replication forks by limiting CDK2 activity.

Camilla Reiter Elbæk, Valdemaras Petrosius, Jan Benada, Louisa Erichsen, Rune Busk Damgaard, Claus Storgaard Sørensen.

  Cellular feedback systems ensure genome maintenance during DNA replication. When replication forks stall, newly replicated DNA is protected by pathways that limit excessive DNA nuclease attacks. Here we show that WEE1 activity guards against nascent DNA degradation at stalled forks. Furthermore, we identify WEE1-dependent suppression of cyclin-dependent kinase 2 (CDK2) as a major activity counteracting fork degradation. We establish DNA2 as the nuclease responsible for excessive fork degradation in WEE1-inhibited cells. In addition, WEE1 appears to be unique among CDK activity suppressors in S phase because neither CHK1 nor p21 promote fork protection as WEE1 does. Our results identify a key role of WEE1 in protecting stalled forks, which is separate from its established role in safeguarding DNA replication initiation. Our findings highlight how WEE1 inhibition evokes massive genome challenges during DNA replication, and this knowledge may improve therapeutic strategies to specifically eradicate cancer cells that frequently harbor elevated DNA replication stress.

Keywords:  CDK; DNA replication; WEE1; cancer; cell cycle; fork protection; genome integrity; nucleases; replication stress

DOI:  https://doi.org/10.1016/j.celrep.2021.110261
Int J Mol Sci. 2022 Jan 14. pii: 893. [Epub ahead of print]23(2):

XRCC1 Prevents Replication Fork Instability during Misincorporation of the DNA Demethylation Bases 5-Hydroxymethyl-2'-Deoxycytidine and 5-Hydroxymethyl-2'-Deoxyuridine.

María José Peña-Gómez, Marina Suárez-Pizarro, Iván V Rosado.

  Whilst avoidance of chemical modifications of DNA bases is essential to maintain genome stability, during evolution eukaryotic cells have evolved a chemically reversible modification of the cytosine base. These dynamic methylation and demethylation reactions on carbon-5 of cytosine regulate several cellular and developmental processes such as embryonic stem cell pluripotency, cell identity, differentiation or tumourgenesis. Whereas these physiological processes are well characterized, very little is known about the toxicity of these cytosine analogues when they incorporate during replication. Here, we report a role of the base excision repair factor XRCC1 in protecting replication fork upon incorporation of 5-hydroxymethyl-2'-deoxycytosine (5hmC) and its deamination product 5-hydroxymethyl-2'-deoxyuridine (5hmU) during DNA synthesis. In the absence of XRCC1, 5hmC exposure leads to increased genomic instability, replication fork impairment and cell lethality. Moreover, the 5hmC deamination product 5hmU recapitulated the genomic instability phenotypes observed by 5hmC exposure, suggesting that 5hmU accounts for the observed by 5hmC exposure. Remarkably, 5hmC-dependent genomic instability and replication fork impairment seen in Xrcc1-/- cells were exacerbated by the trapping of Parp1 on chromatin, indicating that XRCC1 maintains replication fork stability during processing of 5hmC and 5hmU by the base excision repair pathway. Our findings uncover natural epigenetic DNA bases 5hmC and 5hmU as genotoxic nucleosides that threaten replication dynamics and genome integrity in the absence of XRCC1.

Keywords:  5hmU-mediated genomic instability; XRCC1; epigenetic DNA bases; replication fork instability by 5hmC

DOI:  https://doi.org/10.3390/ijms23020893
EMBO J. 2022 Jan 17. e108599

CDK4/6 inhibitors induce replication stress to cause long-term cell cycle withdrawal.

Lisa Crozier, Reece Foy, Brandon L Mouery, Robert H Whitaker, Andrea Corno, Christos Spanos, Tony Ly, Jeanette Gowen Cook, Adrian T Saurin.

  CDK4/6 inhibitors arrest the cell cycle in G1-phase. They are approved to treat breast cancer and are also undergoing clinical trials against a range of other tumour types. To facilitate these efforts, it is important to understand why a cytostatic arrest in G1 causes long-lasting effects on tumour growth. Here, we demonstrate that a prolonged G1 arrest following CDK4/6 inhibition downregulates replisome components and impairs origin licencing. Upon release from that arrest, many cells fail to complete DNA replication and exit the cell cycle in a p53-dependent manner. If cells fail to withdraw from the cell cycle following DNA replication problems, they enter mitosis and missegregate chromosomes causing excessive DNA damage, which further limits their proliferative potential. These effects are observed in a range of tumour types, including breast cancer, implying that genotoxic stress is a common outcome of CDK4/6 inhibition. This unanticipated ability of CDK4/6 inhibitors to induce DNA damage now provides a rationale to better predict responsive tumour types and effective combination therapies, as demonstrated by the fact that CDK4/6 inhibition induces sensitivity to chemotherapeutics that also cause replication stress.

Keywords:  CDK6; Palbociclib; cyclin-dependent kinase; replication stress; senescence

DOI:  https://doi.org/10.15252/embj.2021108599
Nucleic Acids Res. 2022 Jan 17. pii: gkab1255. [Epub ahead of print]

Probing the mechanisms of two exonuclease domain mutators of DNA polymerase ϵ.

Joseph M Dahl, Natalie Thomas, Maxwell A Tracy, Brady L Hearn, Lalith Perera, Scott R Kennedy, Alan J Herr, Thomas A Kunkel.

We report the properties of two mutations in the exonuclease domain of the Saccharomyces cerevisiae DNA polymerase ϵ. One, pol2-Y473F, increases the mutation rate by about 20-fold, similar to the catalytically dead pol2-D290A/E290A mutant. The other, pol2-N378K, is a stronger mutator. Both retain the ability to excise a nucleotide from double-stranded DNA, but with impaired activity. pol2-Y473F degrades DNA poorly, while pol2-N378K degrades single-stranded DNA at an elevated rate relative to double-stranded DNA. These data suggest that pol2-Y473F reduces the capacity of the enzyme to perform catalysis in the exonuclease active site, while pol2-N378K impairs partitioning to the exonuclease active site. Relative to wild-type Pol ϵ, both variants decrease the dNTP concentration required to elicit a switch between proofreading and polymerization by more than an order of magnitude. While neither mutation appears to alter the sequence specificity of polymerization, the N378K mutation stimulates polymerase activity, increasing the probability of incorporation and extension of a mismatch. Considered together, these data indicate that impairing the primer strand transfer pathway required for proofreading increases the probability of common mutations by Pol ϵ, elucidating the association of homologous mutations in human DNA polymerase ϵ with cancer.

DOI: https://doi.org/10.1093/nar/gkab1255
Genes (Basel). 2021 Dec 21. pii: 7. [Epub ahead of print]13(1):

3D Genome Organization: Causes and Consequences for DNA Damage and Repair.

Ànnia Carré-Simon, Emmanuelle Fabre.

  The inability to repair damaged DNA severely compromises the integrity of any organism. In eukaryotes, the DNA damage response (DDR) operates within chromatin, a tightly organized DNA-histone complex in a non-random manner within the nucleus. Chromatin thus orchestrates various cellular processes, including repair. Here, we examine the chromatin landscape before, during, and after the DNA damage, focusing on double strand breaks (DSBs). We study how chromatin is modified during the repair process, not only around the damaged region (in cis), but also genome-wide (in trans). Recent evidence has highlighted a complex landscape in which different chromatin parameters (stiffness, compaction, loops) are transiently modified, defining "codes" for each specific stage of the DDR. We illustrate a novel aspect of DDR where chromatin modifications contribute to the movement of DSB-damaged chromatin, as well as undamaged chromatin, ensuring the mobilization of DSBs, their clustering, and their repair processes.

Keywords:  chromatin dynamics; chromosome organization; double strand break

DOI:  https://doi.org/10.3390/genes13010007
DNA Repair (Amst). 2022 Jan 06. pii: S1568-7864(22)00001-5. [Epub ahead of print]110 103272

Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex.

Michal Dmowski, Malgorzata Jedrychowska, Karolina Makiela-Dzbenska, Milena Denkiewicz-Kruk, Sushma Sharma, Andrei Chabes, Hiroyuki Araki, Iwona J Fijalkowska.

  DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2-7-GINS(Psf1-3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the division labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1-1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1-1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome.

Keywords:  CMG (Cdc45 Mcm2–7 GINS); DNA polymerase delta; DNA polymerase epsilon; DNA replication fidelity; Genome stability; Pol δ; Pol ε; Replication fork

DOI:  https://doi.org/10.1016/j.dnarep.2022.103272
Genes (Basel). 2021 Dec 22. pii: 15. [Epub ahead of print]13(1):

Structural Insights into the Specificity of 8-Oxo-7,8-dihydro-2'-deoxyguanosine Bypass by Family X DNA Polymerases.

Andrea M Kaminski, Thomas A Kunkel, Lars C Pedersen, Katarzyna Bebenek.

  8-oxo-guanine (8OG) is a common base lesion, generated by reactive oxygen species, which has been associated with human diseases such as cancer, aging-related neurodegenerative disorders and atherosclerosis. 8OG is highly mutagenic, due to its dual-coding potential it can pair both with adenine or cytidine. Therefore, it creates a challenge for DNA polymerases striving to correctly replicate and/or repair genomic or mitochondrial DNA. Numerous structural studies provide insights into the mechanistic basis of the specificity of 8OG bypass by DNA polymerases from different families. Here, we focus on how repair polymerases from Family X (Pols β, λ and µ) engage DNA substrates containing the oxidized guanine. We review structures of binary and ternary complexes for the three polymerases, which represent distinct steps in their catalytic cycles-the binding of the DNA substrate and the incoming nucleotide, followed by its insertion and extension. At each of these steps, the polymerase may favor or exclude the correct C or incorrect A, affecting the final outcome, which varies depending on the enzyme.

Keywords:  8-oxo-guanine (8OG); DNA repair; Family X polymerases; base excision repair (BER); nonhomologous end-joining (NHEJ); oxidized base damage

DOI:  https://doi.org/10.3390/genes13010015
Life (Basel). 2022 Jan 13. pii: 108. [Epub ahead of print]12(1):

Transcription-Replication Coordination.

Marco Saponaro.

  Transcription and replication are the two most essential processes that a cell does with its DNA: they allow cells to express the genomic content that is required for their functions and to create a perfect copy of this genomic information to pass on to the daughter cells. Nevertheless, these two processes are in a constant ambivalent relationship. When transcription and replication occupy the same regions, there is the possibility of conflicts between transcription and replication as transcription can impair DNA replication progression leading to increased DNA damage. Nevertheless, DNA replication origins are preferentially located in open chromatin next to actively transcribed regions, meaning that the possibility of conflicts is potentially an accepted incident for cells. Data in the literature point both towards the existence or not of coordination between these two processes to avoid the danger of collisions. Several reviews have been published on transcription-replication conflicts, but we focus here on the most recent findings that relate to how these two processes are coordinated in eukaryotes, considering advantages and disadvantages from coordination, how likely conflicts are at any given time, and which are their potential hotspots in the genome.

Keywords:  DNA damage; DNA replication; G-MiDS; genome instability; transcription; transcription–replication collision

DOI:  https://doi.org/10.3390/life12010108
Eur J Med Chem. 2022 Jan 12. pii: S0223-5234(22)00011-3. [Epub ahead of print]230 114109

Recent advances in DDR (DNA damage response) inhibitors for cancer therapy.

Binbin Cheng, Wei Pan, Yi Xing, Yao Xiao, Jianjun Chen, Zheng Xu.

  DDR (DNA damage response) defects in cells drive tumor formation by promoting DNA mutations, which also provides cancer-specific vulnerabilities that can be targeted by synthetic lethality-based therapies. Until now, PARP inhibitors like olaparib are the first successful case of utilizing synthetic lethality-based therapy to treat cancers with DNA-repairing deficiency (e.g. BRCA1 or BRCA2 mutation), which has fueled the search for more targetable components in the DDR signaling pathway by exploiting synthetic lethality, including but not limited to DNA-PK, ATR, ATM, CHK1, and WEE1. After years of efforts, numerous DDR kinase inhibitors have been discovered. Some of them are being investigated in clinical trials and have shown promising results for cancer therapy. In this review, we summarize the latest advancement in the development of DDR kinase inhibitors including those in preclinical stages and clinical trials, the crystal structures of DDR enzymes, and binding modes of inhibitors with target proteins. The biological functions involving different genes and proteins (ATR, DNA-PK, ATM, PARP, CHK1, and WEE1) are also elucidated.

Keywords:  DNA damage Response; Small molecule inhibitors; Synthetic lethality

DOI:  https://doi.org/10.1016/j.ejmech.2022.114109
Cancers (Basel). 2022 Jan 12. pii: 353. [Epub ahead of print]14(2):

CDK5RAP3, a New BRCA2 Partner That Regulates DNA Repair, Is Associated with Breast Cancer Survival.

Jordi Minguillón, María José Ramírez, Llorenç Rovirosa, Pilar Bustamante-Madrid, Cristina Camps-Fajol, Gorka Ruiz de Garibay, Hermela Shimelis, Helena Montanuy, Roser Pujol, Gonzalo Hernandez, Massimo Bogliolo, Pau Castillo, Penny Soucy, Griselda Martrat, Antonio Gómez, Daniel Cuadras, María J García, Javier Gayarre, Cimba, Conxi Lázaro, Javier Benítez, Fergus J Couch, Miquel Angel Pujana, Jordi Surrallés.

  BRCA2 is essential for homologous recombination DNA repair. BRCA2 mutations lead to genome instability and increased risk of breast and ovarian cancer. Similarly, mutations in BRCA2-interacting proteins are also known to modulate sensitivity to DNA damage agents and are established cancer risk factors. Here we identify the tumor suppressor CDK5RAP3 as a novel BRCA2 helical domain-interacting protein. CDK5RAP3 depletion induced DNA damage resistance, homologous recombination and single-strand annealing upregulation, and reduced spontaneous and DNA damage-induced genomic instability, suggesting that CDK5RAP3 negatively regulates double-strand break repair in the S-phase. Consistent with this cellular phenotype, analysis of transcriptomic data revealed an association between low CDK5RAP3 tumor expression and poor survival of breast cancer patients. Finally, we identified common genetic variations in the CDK5RAP3 locus as potentially associated with breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers. Our results uncover CDK5RAP3 as a critical player in DNA repair and breast cancer outcomes.

Keywords:  BRCA2; CDK5RAP3; DNA repair; breast cancer; chemoresistance

DOI:  https://doi.org/10.3390/cancers14020353
Cancer Lett. 2022 Jan 17. pii: S0304-3835(22)00018-0. [Epub ahead of print]

Disrupting PHF8-TOPBP1 connection elicits a breast tumor-specific vulnerability to chemotherapeutics.

Shuai Ma, Jieyou Zhang, Qiushi Guo, Cheng Cao, Kaiwen Bao, Ling Liu, Charlie Degui Chen, Zhe Liu, Jie Yang, Na Yang, Zhi Yao, Lei Shi.

The DNA damage response (DDR) pathway generally protects against genome instability, and defects in DDR have been exploited therapeutically in cancer treatment. We have reported that histone demethylase PHF8 demethylates TOPBP1 K118 mono-methylation (K118me1) to drive the activation of ATR kinase, one of the master regulators of replication stress. However, whether dysregulation of this physiological signalling is involved in tumorigenesis remains unknown. Here, we showed PHF8-promoted TOPBP1 demethylation is clinically associated with breast tumorigenesis and patient survival. Mammary gland tumors from Phf8 knockout mice grow slowly and exhibit higher level of K118me1, lower ATR activity, and increased chromosomal instability. Importantly, we found that disruption of PHF8-TOPBP1 axis suppresses breast tumorigenesis and creates a breast tumor-specific vulnerability to PARP inhibitor (PARPi) and platinum drug. CRISPR/Cas9 mutation modelling of the deleted or truncated mutation of PHF8 in clinical tumor samples demonstrated breast tumor cells expressing the mimetic variants are more vulnerable to PARPi. Together, our study supports the pursuit of PHF8-TOPBP1 signalling pathway as promising avenues for targeted therapies of PHF8-TOPBP1 proficient tumors, and provides proof-of-concept evidence for loss-of-function of PHF8 as a therapeutic indicator of PARPis.

DOI: https://doi.org/10.1016/j.canlet.2022.01.010
Nucleic Acids Res. 2022 Jan 17. pii: gkab1300. [Epub ahead of print]

53BP1-ACLY-SLBP-coordinated activation of replication-dependent histone biogenesis maintains genomic integrity.

TingTing Wu, Semo Jun, Eun-Ji Choi, Jiao Sun, Eun-Bi Yang, Hyun-Seo Lee, Sang-Yong Kim, Naima Ahmed Fahmi, Qibing Jiang, Wei Zhang, Jeongsik Yong, Jung-Hee Lee, Ho Jin You.

p53-binding protein 1 (53BP1) regulates the DNA double-strand break (DSB) repair pathway and maintains genomic integrity. Here we found that 53BP1 functions as a molecular scaffold for the nucleoside diphosphate kinase-mediated phosphorylation of ATP-citrate lyase (ACLY) which enhances the ACLY activity. This functional association is critical for promoting global histone acetylation and subsequent transcriptome-wide alterations in gene expression. Specifically, expression of a replication-dependent histone biogenesis factor, stem-loop binding protein (SLBP), is dependent upon 53BP1-ACLY-controlled acetylation at the SLBP promoter. This chain of regulation events carried out by 53BP1, ACLY, and SLBP is crucial for both quantitative and qualitative histone biogenesis as well as for the preservation of genomic integrity. Collectively, our findings reveal a previously unknown role for 53BP1 in coordinating replication-dependent histone biogenesis and highlight a DNA repair-independent function in the maintenance of genomic stability through a regulatory network that includes ACLY and SLBP.

DOI: https://doi.org/10.1093/nar/gkab1300
Nat Commun. 2022 Jan 18. 13(1): 359

Deciphering the mechanism of processive ssDNA digestion by the Dna2-RPA ensemble.

Jiangchuan Shen, Yiling Zhao, Nhung Tuyet Pham, Yuxi Li, Yixiang Zhang, Jonathan Trinidad, Grzegorz Ira, Zhi Qi, Hengyao Niu.

Single-stranded DNA (ssDNA) commonly occurs as intermediates in DNA metabolic pathways. The ssDNA binding protein, RPA, not only protects the integrity of ssDNA, but also directs the downstream factor that signals or repairs the ssDNA intermediate. However, it remains unclear how these enzymes/factors outcompete RPA to access ssDNA. Using the budding yeast Saccharomyces cerevisiae as a model system, we find that Dna2 - a key nuclease in DNA replication and repair - employs a bimodal interface to act with RPA both in cis and in trans. The cis-activity makes RPA a processive unit for Dna2-catalyzed ssDNA digestion, where RPA delivers its bound ssDNA to Dna2. On the other hand, activity in trans is mediated by an acidic patch on Dna2, which enables it to function with a sub-optimal amount of RPA, or to overcome DNA secondary structures. The trans-activity mode is not required for cell viability, but is necessary for effective double strand break (DSB) repair.

DOI: https://doi.org/10.1038/s41467-021-27940-y
Proc Natl Acad Sci U S A. 2022 Jan 25. pii: e2119580119. [Epub ahead of print]119(4):

CMG helicase can use ATPγS to unwind DNA: Implications for the rate-limiting step in the reaction mechanism.

Nina Y Yao, Dan Zhang, Olga Yurieva, Michael E O'Donnell.

  The adenosine triphosphate (ATP) analog ATPγS often greatly slows or prevents enzymatic ATP hydrolysis. The eukaryotic CMG (Cdc45, Mcm2 to 7, GINS) replicative helicase is presumed unable to hydrolyze ATPγS and thus unable to perform DNA unwinding, as documented for certain other helicases. Consequently, ATPγS is often used to "preload" CMG onto forked DNA substrates without unwinding before adding ATP to initiate helicase activity. We find here that CMG does hydrolyze ATPγS and couples it to DNA unwinding. Indeed, the rate of unwinding of a 20- and 30-mer duplex fork of different sequences by CMG is only reduced 1- to 1.5-fold using ATPγS compared with ATP. These findings imply that a conformational change is the rate-limiting step during CMG unwinding, not hydrolysis. Instead of using ATPγS for loading CMG onto DNA, we demonstrate here that nonhydrolyzable adenylyl-imidodiphosphate (AMP-PNP) can be used to preload CMG onto a forked DNA substrate without unwinding.

Keywords:  ATPgammaS; CMG helicase; DNA replication; rate-limiting step; staircase model

DOI:  https://doi.org/10.1073/pnas.2119580119
Nucleic Acids Res. 2022 Jan 21. pii: gkac009. [Epub ahead of print]

TOPORS-mediated RAD51 SUMOylation facilitates homologous recombination repair.

Gurusamy Hariharasudhan, Seo-Yeon Jeong, Min-Ji Kim, Sung Mi Jung, Gwanwoo Seo, Ju-Ran Moon, Sumi Lee, In-Youb Chang, Younghoon Kee, Ho Jin You, Jung-Hee Lee.

Homologous recombination (HR) is critical for error-free repair of DNA double-strand breaks. Chromatin loading of RAD51, a key protein that mediates the recombination, is a crucial step in the execution of the HR repair. Here, we present evidence that SUMOylation of RAD51 is crucial for the RAD51 recruitment to chromatin and HR repair. We found that topoisomerase 1-binding arginine/serine-rich protein (TOPORS) induces the SUMOylation of RAD51 at lysine residues 57 and 70 in response to DNA damaging agents. The SUMOylation was facilitated by an ATM-induced phosphorylation of TOPORS at threonine 515 upon DNA damage. Knockdown of TOPORS or expression of SUMOylation-deficient RAD51 mutants caused reduction in supporting normal RAD51 functions during the HR repair, suggesting the physiological importance of the modification. We found that the SUMOylation-deficient RAD51 reduces the association with its crucial binding partner BRCA2, explaining its deficiency in supporting the HR repair. These findings altogether demonstrate a crucial role for TOPORS-mediated RAD51 SUMOylation in promoting HR repair and genomic maintenance.

DOI: https://doi.org/10.1093/nar/gkac009
Nat Commun. 2022 Jan 18. 13(1): 360

53BP1 regulates heterochromatin through liquid phase separation.

Lei Zhang, Xinran Geng, Fangfang Wang, Jinshan Tang, Yu Ichida, Arishya Sharma, Sora Jin, Mingyue Chen, Mingliang Tang, Franklin Mayca Pozo, Wenxiu Wang, Janet Wang, Michal Wozniak, Xiaoxia Guo, Masaru Miyagi, Fulai Jin, Yongjie Xu, Xinsheng Yao, Youwei Zhang.

Human 53BP1 is primarily known as a key player in regulating DNA double strand break (DSB) repair choice; however, its involvement in other biological process is less well understood. Here, we report a previously uncharacterized function of 53BP1 at heterochromatin, where it undergoes liquid-liquid phase separation (LLPS) with the heterochromatin protein HP1α in a mutually dependent manner. Deletion of 53BP1 results in a reduction in heterochromatin centers and the de-repression of heterochromatic tandem repetitive DNA. We identify domains and residues of 53BP1 required for its LLPS, which overlap with, but are distinct from, those involved in DSB repair. Further, 53BP1 mutants deficient in DSB repair, but proficient in LLPS, rescue heterochromatin de-repression and protect cells from stress-induced DNA damage and senescence. Our study suggests that in addition to DSB repair modulation, 53BP1 contributes to the maintenance of heterochromatin integrity and genome stability through LLPS.

DOI: https://doi.org/10.1038/s41467-022-28019-y
Sci Adv. 2022 Jan 21. 8(3): eabg6383

Interactions with stromal cells promote a more oxidized cancer cell redox state in pancreatic tumors.

Rupsa Datta, Sharanya Sivanand, Allison N Lau, Logan V Florek, Anna M Barbeau, Jeffrey Wyckoff, Melissa C Skala, Matthew G Vander Heiden.

Access to electron acceptors supports oxidized biomass synthesis and can be limiting for cancer cell proliferation, but how cancer cells overcome this limitation in tumors is incompletely understood. Nontransformed cells in tumors can help cancer cells overcome metabolic limitations, particularly in pancreatic cancer, where pancreatic stellate cells (PSCs) promote cancer cell proliferation and tumor growth. However, whether PSCs affect the redox state of cancer cells is not known. By taking advantage of the endogenous fluorescence properties of reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide cofactors we use optical imaging to assess the redox state of pancreatic cancer cells and PSCs and find that direct interactions between PSCs and cancer cells promote a more oxidized state in cancer cells. This suggests that metabolic interaction between cancer cells and PSCs is a mechanism to overcome the redox limitations of cell proliferation in pancreatic cancer.

DOI: https://doi.org/10.1126/sciadv.abg6383
Cancer Res. 2022 Jan 19. pii: canres.2229.2021. [Epub ahead of print]

Targeting squalene epoxidase interrupts homologous recombination via the ER stress response and promotes radiotherapy efficacy.

Zhipeng Hong, Tao Liu, Lingfeng Wan, Pengyan Fa, Pankaj Kumar, Yanan Cao, Chandra Bhushan Prasad, Zhaojun Qiu, Joseph Liu, Hongbing Wang, Zaibo Li, Qi-En Wang, Peixuan Guo, Deliang Guo, Ayse S Yilmaz, Lanchun Lu, Ioanna Papandreou, Naduparambil K Jacob, Chunhong Yan, Xiaoli Zhang, Qing-Bai She, Zhefu Ma, Junran Zhang.

Over 50% of all cancer patients are treated with radiation therapy (RT). However, RT is often insufficient as a monotherapy and requires a non-toxic radiosensitizer. Squalene epoxidase (SQLE) controls cholesterol biosynthesis by converting squalene to 2,3-oxidosqualene. Given that SQLE is frequently overexpressed in human cancer, this study investigated the importance of SQLE in breast cancer (BC) and non-small cell lung cancer (NSCLC), two cancers often treated with RT. SQLE-positive immunohistochemical staining was observed in 68% of BC and 56% of NSCLC specimens versus 15% and 25% in normal breast and lung tissue, respectively. Importantly, SQLE expression was an independent predictor of poor prognosis, and pharmacological inhibition of SQLE enhanced breast and lung cancer cell radiosensitivity. In addition, SQLE inhibition enhanced sensitivity to PARP inhibition. Inhibition of SQLE interrupted homologous recombination by suppressing ATM activity via the translational upregulation of wild-type p53-induced phosphatase (WIP1), regardless of the p53 status. SQLE inhibition and subsequent squalene accumulation promoted this upregulation by triggering the endoplasmic reticulum (ER) stress response. Collectively, these results identify a novel tumor-specific radiosensitizer by revealing unrecognized crosstalk between squalene metabolites, ER stress, and the DNA damage response (DDR). Although SQLE inhibitors have been used as antifungal agents in the clinic, they have not yet been used as antitumor agents. Repurposing existing SQLE-inhibiting drugs may provide new cancer treatments.

DOI: https://doi.org/10.1158/0008-5472.CAN-21-2229
Sci Rep. 2022 Jan 19. 12(1): 974

Pre-clinical activity of the oral DNA-PK inhibitor, peposertib (M3814), combined with radiation in xenograft models of cervical cancer.

Sushmita B Gordhandas, Beryl Manning-Geist, Christina Henson, Gopa Iyer, Ginger J Gardner, Yukio Sonoda, Kathleen N Moore, Carol Aghajanian, M Herman Chui, Rachel N Grisham.

DNA-dependent protein kinase (DNA-PK) plays a crucial role in repair of DNA double-strand breaks by facilitating non-homologous end-joining. Inhibitors of DNA-PK have the potential to block DNA repair and enhance DNA-damaging agents. Peposertib (M3814) is a DNA-PK inhibitor that has shown preclinical activity in combination with DNA-damaging agents, including ionizing radiation (IR) and topoisomerase II inhibitors. Here we evaluated the activity of peposertib (M3814) in combination with radiation in a mouse xenograft model of HPV-associated cervical cancer. Athymic nude female mice with established tumors derived from HeLa cells injected into the flank were treated with vehicle alone (n = 3), IR alone (n = 4), and peposertib (M38814) in combination with IR (M3814 + IR; n = 4). While IR alone was associated with a trend towards decreased tumor volume compared with untreated, only the M3814 + IR treatment arm was associated with consistent and significant reduction in tumor burden, which correlated with higher levels of γ-H2AX in tumor cells, a marker of double-strand DNA breaks. Our data support further clinical evaluation of the combination of peposertib (M38814) and IR in cervical cancer.

DOI: https://doi.org/10.1038/s41598-021-04618-5
Nucleic Acids Res. 2022 Jan 21. pii: gkac023. [Epub ahead of print]

POLIE suppresses telomerase-mediated telomere G-strand extension and helps ensure proper telomere C-strand synthesis in trypanosomes.

M A G Rabbani, Maiko Luis Tonini, Marjia Afrin, Bibo Li.

Trypanosoma brucei causes human African trypanosomiasis and sequentially expresses distinct VSGs, its major surface antigen, to achieve host immune evasion. VSGs are monoallelically expressed from subtelomeric loci, and telomere proteins regulate VSG monoallelic expression and VSG switching. T. brucei telomerase is essential for telomere maintenance, but no regulators of telomerase have been identified. T. brucei appears to lack OB fold-containing telomere-specific ssDNA binding factors that are critical for coordinating telomere G- and C-strand syntheses in higher eukaryotes. We identify POLIE as a telomere protein essential for telomere integrity. POLIE-depleted cells have more frequent VSG gene conversion-mediated VSG switching and an increased amount of telomeric circles (T-circles), indicating that POLIE suppresses DNA recombination at the telomere/subtelomere. POLIE-depletion elongates telomere 3' overhangs dramatically, indicating that POLIE is essential for coordinating DNA syntheses of the two telomere strands. POLIE depletion increases the level of telomerase-dependent telomere G-strand extension, identifying POLIE as the first T. brucei telomere protein that suppresses telomerase. Furthermore, depletion of POLIE results in an elevated telomeric C-circle level, suggesting that the telomere C-strand experiences replication stress and that POLIE may promote telomere C-strand synthesis. Therefore, T. brucei uses a novel mechanism to coordinate the telomere G- and C-strand DNA syntheses.

DOI: https://doi.org/10.1093/nar/gkac023
Nucleic Acids Res. 2022 Jan 21. pii: gkac005. [Epub ahead of print]

The fork protection complex recruits FACT to reorganize nucleosomes during replication.

Barbara Safaric, Erika Chacin, Matthias J Scherr, Lional Rajappa, Christian Gebhardt, Christoph F Kurat, Thorben Cordes, Karl E Duderstadt.

Chromosome replication depends on efficient removal of nucleosomes by accessory factors to ensure rapid access to genomic information. Here, we show this process requires recruitment of the nucleosome reorganization activity of the histone chaperone FACT. Using single-molecule FRET, we demonstrate that reorganization of nucleosomal DNA by FACT requires coordinated engagement by the middle and C-terminal domains of Spt16 and Pob3 but does not require the N-terminus of Spt16. Using structure-guided pulldowns, we demonstrate instead that the N-terminal region is critical for recruitment by the fork protection complex subunit Tof1. Using in vitro chromatin replication assays, we confirm the importance of these interactions for robust replication. Our findings support a mechanism in which nucleosomes are removed through the coordinated engagement of multiple FACT domains positioned at the replication fork by the fork protection complex.

DOI: https://doi.org/10.1093/nar/gkac005
Leukemia. 2022 Jan 17.

HMCES safeguards genome integrity and long-term self-renewal of hematopoietic stem cells during stress responses.

Yinghao Pan, Hongna Zuo, Fei Wen, Fei Huang, Yezhang Zhu, Lanrui Cao, Qian-Qian Sha, Yang Li, Huiying Zhang, Miao Shi, Chengzhen Liang, Jun Huang, Lin Zou, Heng-Yu Fan, Zhenyu Ju, Hu Wang, Li Shen.

Hematopoietic stress drives quiescent hematopoietic stem cells (HSCs) to proliferate, generating reactive oxygen species (ROS) and oxidative DNA damage including abasic sites. Such a coupling between rapid DNA replication and a burst of abasic site formation during HSC stress responses, however, presents a challenge to accurately repair abasic sites located in replication-associated single-stranded DNA. Here we show that HMCES, a novel shield of abasic sites, plays pivotal roles in overcoming this challenge upon HSC activation. While HMCES was dispensable for steady-state hematopoiesis, Hmces-deficient HSCs exhibited compromised long-term self-renewal capacity in response to hematopoietic stress such as myeloablation and transplantation. Loss of HMCES resulted in accumulation of DNA lesions due to impaired resolution of abasic sites generated by activation-induced ROS in activated HSCs and broad downregulation of DNA damage response and repair pathways. Moreover, Hmces-deficient mice died from bone marrow failure after exposure to sublethal irradiation, which also produces ROS. Notably, dysregulation of HMCES occurs frequently in acute lymphocytic leukemia (ALL) and is associated with poor clinical outcomes. Together, our findings not only highlighted HMCES as a novel genome protector in activated HSCs, but also position it as a potential selective target against ALL while sparing normal hematopoiesis.

DOI: https://doi.org/10.1038/s41375-021-01499-5
Cell Rep. 2022 Jan 18. pii: S2211-1247(21)01793-9. [Epub ahead of print]38(3): 110278

Lineage-specific silencing of PSAT1 induces serine auxotrophy and sensitivity to dietary serine starvation in luminal breast tumors.

Bo-Hyun Choi, Vipin Rawat, Jenny Högström, Philippa A Burns, Kelly O Conger, Mete Emir Ozgurses, Jaymin M Patel, Tejas S Mehta, Angelica Warren, Laura M Selfors, Taru Muranen, Jonathan L Coloff.

  A major challenge of targeting metabolism for cancer therapy is pathway redundancy, in which multiple sources of critical nutrients can limit the effectiveness of some metabolism-targeted therapies. Here, we analyze lineage-dependent gene expression in human breast tumors to identify differences in metabolic gene expression that may limit pathway redundancy and create therapeutic vulnerabilities. We find that the serine synthesis pathway gene PSAT1 is the most depleted metabolic gene in luminal breast tumors relative to basal tumors. Low PSAT1 prevents de novo serine biosynthesis and sensitizes luminal breast cancer cells to serine and glycine starvation in vitro and in vivo. This PSAT1 expression disparity preexists in the putative cells of origin of basal and luminal tumors and is due to luminal-specific hypermethylation of the PSAT1 gene. Our data demonstrate that luminal breast tumors are auxotrophic for serine and may be uniquely sensitive to therapies targeting serine availability.

Keywords:  PHGDH; PSAT1; auxotrophy; breast cancer; diet; luminal tumors; serine; tumor metabolism

DOI:  https://doi.org/10.1016/j.celrep.2021.110278
Cancer Treat Rev. 2022 Jan 05. pii: S0305-7372(21)00185-7. [Epub ahead of print]104 102337

Therapeutic implications of germline vulnerabilities in DNA repair for precision oncology.

Shreya M Shah, Elena V Demidova, Randy W Lesh, Michael J Hall, Mary B Daly, Joshua E Meyer, Martin J Edelman, Sanjeevani Arora.

  DNA repair vulnerabilities are present in a significant proportion of cancers. Specifically, germline alterations in DNA repair not only increase cancer risk but are associated with treatment response and clinical outcomes. The therapeutic landscape of cancer has rapidly evolved with the FDA approval of therapies that specifically target DNA repair vulnerabilities. The clinical success of synthetic lethality between BRCA deficiency and poly(ADP-ribose) polymerase (PARP) inhibition has been truly revolutionary. Defective mismatch repair has been validated as a predictor of response to immune checkpoint blockade associated with durable responses and long-term benefit in many cancer patients. Advances in next generation sequencing technologies and their decreasing cost have supported increased genetic profiling of tumors coupled with germline testing of cancer risk genes in patients. The clinical adoption of panel testing for germline assessment in high-risk individuals has generated a plethora of genetic data, particularly on DNA repair genes. Here, we highlight the therapeutic relevance of germline aberrations in DNA repair to identify patients eligible for precision treatments such as PARP inhibitors (PARPis), immune checkpoint blockade, chemotherapy, radiation therapy and combined treatment. We also discuss emerging mechanisms that regulate DNA repair.

Keywords:  DNA repair; Germline; Immune checkpoint inhibitors; PARP inhibitors; Precision oncology; Therapeutic response

DOI:  https://doi.org/10.1016/j.ctrv.2021.102337
Sci Adv. 2022 Jan 21. 8(3): eabj8357

SF3B1 homeostasis is critical for survival and therapeutic response in T cell leukemia.

Cuijuan Han, Alireza Khodadadi-Jamayran, Adam H Lorch, Qi Jin, Valentina Serafin, Ping Zhu, Yuliya Politanska, Limin Sun, Blanca T Gutierrez-Diaz, Marina V Pryzhkova, Hiam Abdala-Valencia, Elizabeth Thomas Bartom, Barbara Buldini, Giuseppe Basso, Sadanandan E Velu, Kavitha Sarma, Basil B Mattamana, Byoung-Kyu Cho, Rebecca C Obeng, Young Ah Goo, Philip W Jordan, Aristotelis Tsirigos, Yalu Zhou, Panagiotis Ntziachristos.

The production of noncanonical mRNA transcripts is associated with cell transformation. Driven by our previous findings on the sensitivity of T cell acute lymphoblastic leukemia (T-ALL) cells to SF3B1 inhibitors, we identified that SF3B1 inhibition blocks T-ALL growth in vivo with no notable associated toxicity. We also revealed protein stabilization of the U2 complex component SF3B1 via deubiquitination. Our studies showed that SF3B1 inhibition perturbs exon skipping, leading to nonsense-mediated decay and diminished levels of DNA damage response-related transcripts, such as the serine/threonine kinase CHEK2, and impaired DNA damage response. We also identified that SF3B1 inhibition leads to a general decrease in R-loop formation. We further demonstrate that clinically used SF3B1 inhibitors synergize with CHEK2 inhibitors and chemotherapeutic drugs to block leukemia growth. Our study provides the proof of principle for posttranslational regulation of splicing components and associated roles and therapeutic implications for the U2 complex in T cell leukemia.

DOI: https://doi.org/10.1126/sciadv.abj8357
Nucleic Acids Res. 2022 Jan 20. pii: gkac011. [Epub ahead of print]

TRIM21 suppresses CHK1 activation by preferentially targeting CLASPIN for K63-linked ubiquitination.

Xuefei Zhu, Jingwei Xue, Xing Jiang, Yamin Gong, Congwen Gao, Ting Cao, Qian Li, Lulu Bai, Yuwei Li, Gaixia Xu, Bin Peng, Xingzhi Xu.

Expression of the E3 ligase TRIM21 is increased in a broad spectrum of cancers; however, the functionally relevant molecular pathway targeted by TRIM21 overexpression remains largely unknown. Here, we show that TRIM21 directly interacts with and ubiquitinates CLASPIN, a mediator for ATR-dependent CHK1 activation. TRIM21-mediated K63-linked ubiquitination of CLASPIN counteracts the K6-linked ubiquitination of CLASPIN which is essential for its interaction with TIPIN and subsequent chromatin loading. We further show that overexpression of TRIM21, but not a TRIM21 catalytically inactive mutant, compromises CHK1 activation, leading to replication fork instability and tumorigenesis. Our findings demonstrate that TRIM21 suppresses CHK1 activation by preferentially targeting CLASPIN for K63-linked ubiquitination, providing a potential target for cancer therapy.

DOI: https://doi.org/10.1093/nar/gkac011
SLAS Discov. 2022 Jan 08. pii: S2472-5552(21)00025-3. [Epub ahead of print]

Validation of a high throughput screening assay to identify small molecules that target the eukaryotic replicative helicase.

Jordan Sanders, Michael Castiglione, Tongying Shun, Laura L Vollmer, Mark E Schurdak, Andreas Vogt, Anthony Schwacha.

  Mcm2-7 is the catalytic core of the eukaryotic replicative helicase, which together with CDC45 and the GINS complex unwind parental DNA to generate templates for DNA polymerase. Being a highly regulated and complex enzyme that operates via an incompletely understood multi-step mechanism, molecular probes of Mcm2-7 that interrogate specific mechanistic steps would be useful tools for research and potential future chemotherapy. Based upon a synthetic lethal approach, we previously developed a budding yeast multivariate cell-based high throughput screening (HTS) assay to identify putative Mcm inhibitors by their ability to specifically cause a growth defect in an mcm mutant relative to a wild-type strain[1]. Here, as proof of concept, we used this assay to screen a 1280-member compound library (LOPAC) for potential Mcm2-7 inhibitors. Primary screening and dose-dependent retesting identified twelve compounds from this library that specifically inhibited the growth of the Mcm mutant relative to the corresponding wild-type strain (0.9 % hit rate). Secondary assays were employed to rule out non-specific DNA damaging agents, establish direct protein-ligand interaction via biophysical methods, and verify in vivo DNA replication inhibition via fluorescence activated cell sorter analysis (FACS). We identified one agent (β-carboline-3-carboxylic acid N-methylamide, CMA) that physically bound to the purified Mcm2-7 complex (Kdapp119 µM), and at slightly higher concentrations specifically blocked S-phase cell cycle progression of the wild-type strain. In total, identification of Mcm2-7 as a CMA target validates our synthetic lethal HTS assay paradigm as a tool to identify chemical probes for the Mcm2-7 replicative helicase.

Keywords:  DNA replication; HTS assay development; Mcm2–7; Multivariate analysis; Yeast screening technology

DOI:  https://doi.org/10.1016/j.slasd.2021.12.006
Pharmaceuticals (Basel). 2021 Dec 28. pii: 37. [Epub ahead of print]15(1):

BI-2536 Promotes Neuroblastoma Cell Death via Minichromosome Maintenance Complex Components 2 and 10.

Chiao-Hui Hsieh, Hsiang-Ning Yeh, Chen-Tsung Huang, Wei-Hsuan Wang, Wen-Ming Hsu, Hsuan-Cheng Huang, Hsueh-Fen Juan.

  DNA replication is initiated with the recognition of the starting point of multiple replication forks by the origin recognition complex and activation of the minichromosome maintenance complex 10 (MCM10). Subsequently, DNA helicase, consisting of the MCM protein subunits MCM2-7, unwinds double-stranded DNA and DNA synthesis begins. In previous studies, replication factors have been used as clinical targets in cancer therapy. The results showed that MCM2 could be a proliferation marker for numerous types of malignant cancer. We analyzed samples obtained from patients with neuroblastoma, revealing that higher levels of MCM2 and MCM10 mRNA were associated with poor survival rate. Furthermore, we combined the results of the perturbation-induced reversal effects on the expression levels of MCM2 and MCM10 and the sensitivity correlation between perturbations and MCM2 and MCM10 from the Cancer Therapeutics Response Portal database. Small molecule BI-2536, a polo-like kinase 1 (PLK-1) inhibitor, is a candidate for the inhibition of MCM2 and MCM10 expression. To test this hypothesis, we treated neuroblastoma cells with BI-2536. The results showed that the drug decreased cell viability and reduced the expression levels of MCM2 and MCM10. Functional analysis further revealed enrichments of gene sets involved in mitochondria, cell cycle, and DNA replication for BI-2536-perturbed transcriptome. We used cellular assays to demonstrate that BI-2536 promoted mitochondria fusion, G2/M arrest, and apoptosis. In summary, our findings provide a new strategy for neuroblastoma therapy with BI-2536.

Keywords:  BI-2536; cell death; minichromosome maintenance complex 2 and 10; mitochondria fusion; neuroblastoma

DOI:  https://doi.org/10.3390/ph15010037
Oncogene. 2022 Jan 20.

Targeting glutamine metabolism network for the treatment of therapy-resistant prostate cancer.

Lingfan Xu, Bing Zhao, William Butler, Huan Xu, Nan Song, Xufeng Chen, J Spencer Hauck, Xia Gao, Hong Zhang, Jeff Groth, Qing Yang, Yue Zhao, David Moon, Daniel George, Yinglu Zhou, Yiping He, Jiaoti Huang.

Advanced and aggressive prostate cancer (PCa) depends on glutamine for survival and proliferation. We have previously shown that inhibition of glutaminase 1, which catalyzes the rate-limiting step of glutamine catabolism, achieves significant therapeutic effect; however, therapy resistance is inevitable. Here we report that while the glutamine carbon is critical to PCa survival, a parallel pathway of glutamine nitrogen catabolism that actively contributes to pyrimidine assembly is equally important for PCa cells. Importantly, we demonstrate a reciprocal feedback mechanism between glutamine carbon and nitrogen pathways which leads to therapy resistance when one of the two pathways is inhibited. Combination treatment to inhibit both pathways simultaneously yields better clinical outcome for advanced PCa patients.

DOI: https://doi.org/10.1038/s41388-021-02155-z
J Fungi (Basel). 2021 Dec 29. pii: 29. [Epub ahead of print]8(1):

Purine Auxotrophic Starvation Evokes Phenotype Similar to Stationary Phase Cells in Budding Yeast.

Agnese Kokina, Kristel Tanilas, Zane Ozolina, Karlis Pleiko, Karlis Shvirksts, Ilze Vamza, Janis Liepins.

  Purine auxotrophy is an abundant trait among eukaryotic parasites and a typical marker for many budding yeast strains. Supplementation with an additional purine source (such as adenine) is necessary to cultivate these strains. If not supplied in adequate amounts, purine starvation sets in. We explored purine starvation effects in a model organism, a budding yeast Saccharomyces cerevisiae ade8 knockout, at the level of cellular morphology, central carbon metabolism, and global transcriptome. We observed that purine-starved cells stopped their cycle in G1/G0 state and accumulated trehalose, and the intracellular concentration of AXP decreased, but adenylate charge remained stable. Cells became tolerant to severe environmental stresses. Intracellular RNA concentration decreased, and massive downregulation of ribosomal biosynthesis genes occurred. We proved that the expression of new proteins during purine starvation is critical for cells to attain stress tolerance phenotype Msn2/4p targets are upregulated in purine-starved cells when compared to cells cultivated in purine-rich media. The overall transcriptomic response to purine starvation resembles that of stationary phase cells. Our results demonstrate that the induction of a strong stress resistance phenotype in budding yeast can be caused not only by natural starvation, but also starvation for metabolic intermediates, such as purines.

Keywords:  Saccharomyces cerevisiae; purines; starvation; stress resistance

DOI:  https://doi.org/10.3390/jof8010029
Eur J Haematol. 2022 Jan 18.

Treatment Outcomes with Purine Nucleoside Analog Alone or with Rituximab for Hairy Cell Leukemia at First Relapse.

Rachel Hu, Wei Wei, Agrima Mian, Kristen Gonter-Aubin, Charlene Kabel, Anthony Mato, Deborah M Stephens, Ashley Hanlon, Sirin Khajavian, Mazyar Shadman, Danielle Brander, Yazan Madanat, Jae H Park, Martin Tallman, Javier Pinilla-Ibarz, Brian T Hill.

  INTRODUCTION: Frontline treatment of hairy cell leukemia (HCL) with a single course of the purine nucleoside analog (PNA) produces a high rate of complete remission (CR) with prolonged durations. At the time of relapse, although treatment guidelines recommend re-treatment with a PNA alone or in combination with rituximab (R), practice patterns vary and data supporting each approach are limited.METHODS: We conducted a multi-site outcomes analysis of patients treated for HCL between 1995-2018 at 6 US medical centers. All patients were treated with frontline PNA and subsequently required treatment with a PNA alone (PNA) or with R (+R).
RESULTS: Of the 88 patients analyzed, 56 (63.6%) received second line PNA and 22 (36.4%) received a PNA+R. Baseline characteristics of both groups were similar. There was no difference in median PFS [67 months (95% CI 43.8-non reached (NR)) vs. 65 months (95% CI 60-NR)] or 5 year OS [98% (95% CI 0.94-1) vs. 94% (95% CI 0.83-1), P=0.104] in the PNA vs. PNA+R cohorts, respectively.
CONCLUSION: To our knowledge, this is the largest study evaluating the role of R in treatment of relapsed HCL and suggests that there is no advantage to the addition of R to PNA therapy at the time of first re-treatment.

Keywords:  Hairy cell leukemia; purine nucleoside analog; rituximab

DOI:  https://doi.org/10.1111/ejh.13744