Biology subjects

Campbell, C. S.

Publications and source records attributed to Campbell, C. S..

3 recordsLinked to original sources

bioRxiv2022

Adaptation to spindle assembly checkpoint inhibition through the selection of specific aneuploidies

Both the presence of an abnormal complement of chromosomes (aneuploidy) and an increased frequency of chromosome missegregation (chromosomal instability) are hallmarks of cancer. Analyses of cancer genome data have identified certain aneuploidy patterns in tumors; however, the bases behind their selection are largely unexplored. By establishing time-resolved long-term adaptation protocols, we found that human cells adapt to persistent spindle assembly checkpoint (SAC) inhibition by acquiring specific chromosome arm gains and losses. Independently adapted populations converge on complex karyotypes, which over time are refined to contain ever smaller chromosomal changes. Of note, the frequencies of chromosome arm gains in adapted cells correlate very well with those detected in cancers, suggesting that our cellular adaptation approach recapitulates selective traits that dictate pan-cancer aneuploidy patterns. We further engineered specific aneuploidies to determine the genetic basis behind the observed karyotype patterns. These experiments demonstrated that the adapted and engineered aneuploid cell lines limit CIN by extending mitotic duration. Heterozygous deletions of key SAC and APC/C genes recapitulated the rescue phenotypes of the monosomic chromosomes. We conclude that aneuploidy-induced gene dosage imbalances of individual mitotic regulators are sufficient for altering mitotic timing to reduce CIN.

cell biology↗

bioRxiv2022

Multiple routes of adaptation to high levels of CIN and aneuploidy in budding yeast

Both an increased frequency of chromosome missegregation (chromosomal instability) and the presence of an abnormal complement of chromosomes (aneuploidy) are hallmarks of cancer. Paradoxically, both chromosomal instability and aneuploidy are also associated with substantial decreases in cellular fitness. To better understand how cells are able to adapt to high levels of chromosomal instability, we previously examined yeast cells that were deleted of the gene BIR1, a member of the chromosomal passenger complex (CPC). The CPC is an essential regulator of chromosome segregation fidelity. We found bir1{Delta} cells quickly adapted by acquiring specific combinations of beneficial aneuploidies. However, targeted mutations of specific genes were notably absent in the short term. In this study, we monitored these yeast strains for longer periods of time to determine how cells adapt to high levels of both CIN and aneuploidy in the long term. We identify suppressor mutations that mitigate the chromosome missegregation phenotype. The mutated proteins fall into four main categories: outer kinetochore subunits, members of the SCFCdc4 complex, the mitotic kinase Mps1, and a member of the CPC itself. These mutants function in two distinct ways, as mutations in the outer kinetochore suppress Bir1 deletion indirectly by destabilizing connections between the chromosomes and the mitotic spindle, whereas the other three categories of mutations affect the CPC directly. As a consequence of the accumulation of suppressor point mutations, overall levels of aneuploidy decreased. These experiments demonstrate a timeline of adaptation to high rates of CIN wherein cells first acquire specific aneuploidies that suppress the CIN phenotype, next develop point mutations that more specifically target the source of CIN, and finally reduce the level of aneuploidy to relieve the fitness burden placed by aneuploidy on the cell.

cell biology↗

bioRxiv2021

Inhibition of Heat Shock Protein 90 in the Spinal Cord Improves the Therapeutic Index of Morphine

Opioid drugs like morphine are the gold standard for the treatment of chronic pain, but are limited by adverse side effects, such as tolerance, constipation, and reward/addiction. In our earlier work, we showed that Heat shock protein 90 (Hsp90) has a crucial role in regulating opioid signaling that differs between brain and spinal cord; Hsp90 inhibition in brain blocks opioid pain relief, while inhibition in the spinal cord enhances it. Building on these findings here, we injected the non-selective Hsp90 inhibitor KU-32 directly into the spinal cord of male and female CD-1 mice, showing that morphine anti-nociceptive potency was boosted by 1.9-3.5 fold in the pain models of tail flick, post-surgical paw incision, and HIV peripheral neuropathy. At the same time, morphine tolerance was reduced from 21 fold to 2.9 fold and established tolerance was rescued, while the potency of constipation and reward (as measured by conditioned place preference) was unchanged. These results demonstrate that spinal Hsp90 inhibition can improve the therapeutic index of morphine. However, we also found that systemic non-selective Hsp90 inhibition resulted in a brain-like effect, blocking opioid pain relief. We thus sought a way to circumvent the effects of brain Hsp90 inhibition by investigating the molecular Hsp90 isoforms active in regulating opioid signaling in both regions. Using selective small molecule inhibitors and CRISPR gene editing, we found that 3 Hsp90 isoforms regulated spinal cord opioid signaling (Hsp90, Hsp90{beta}, and Grp94) while our previous work showed only Hsp90 was active in brain. We thus hypothesized that a systemically delivered selective inhibitor to Hsp90{beta} or Grp94 could selectively inhibit spinal cord Hsp90 activity, resulting in enhanced opioid pain relief and decreased side effects. We tested this hypothesis using intravenous delivery of KUNB106 (Hsp90{beta}) and KUNG65 (Grp94), showing that both drugs enhanced morphine potency in tail flick and paw incision pain while rescuing anti-nociceptive tolerance. We also found that intravenous KUNA115 (Hsp90) fully blocked morphine anti-nociception. Together, these results suggest that selective inhibition of spinal cord Hsp90 isoforms is a novel, translationally feasible strategy to improve the therapeutic index of opioids.

neuroscience↗