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PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
While restricted to specialized cell types in normal human tissues, polyploidy has been associated with almost all solid and liquid cancers .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Interestingly, chemotherapy, the standard treatment for Diffuse Large B-Cell Lymphoma (DLBCL) has been observed to increase ploidy over time, a known mechanism for drug resistance [, , , , ].
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
These polyploid cells can avoid apoptosis and/or escape cell death though cellular senescence .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
However, post-chemotherapy polyploid cells have been shown to re-enter the cell cycle, resulting in relapsed disease .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Chemotherapy induced polyploidy (CIP) is a mechanism of inherited drug resistance.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Additionally, proliferating CIP cancer cells can produce progeny with increased ploidy or aneuploid genomes that drive aggressive disease .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
CIP can arise in a variety of ways, including cell cycle slippage, an unconventional cell cycle where DNA replication is uncoupled from mitosis .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
These cancer cells post-mitosis will begin replicate their DNA during the synthesis (S) phase and then transition to a gap (G2) phase, thereby skipping G1 entirely .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Alternatively, cells can undergo mitotic slippage by entering mitosis, arresting, reentering G1 phase without attempting or completing cytokinesis (endomitosis) .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
CIP cells may double their genome or, conversely, produce daughter cells with smaller aneuploid genomes due to the increased risk of chromosomal instability (CIN) and chromosome loss during mitosis .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
A myriad of aberrant conditions can contribute to CIN, including the presence of multiple centrosomes, defects in proper kinetochore–microtubule attachment, spindle assembly checkpoint (SAC) dysfunction, defects in chromosome cohesion, nuclear envelope reformation, and cytokinesis [, , , ].
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
The Aurora Kinase (AK) family of mitotic enzymes has been identified as a major proponent of suppressing chromosome mis-segregation .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Of interest these c-MYC-regulated mitotic serine/threonine kinases, AK-A and AK-B, induce tetraploidy/polyploidy in DLBCL when disrupted .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
AK-A is associated with centrosome duplication and mitotic spindle formation, while AK-B (chromosome passenger protein) localizes to the prometaphase kinetochores and the spindle midzone of anaphase cells .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
In many tumor types, inhibitors of AK-A and/or AK-B lead to polyploidy, latter due to cytokinesis failure .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
AK-A inhibition mediated polyploid tumor cells appear resistant to a range of chemotherapies, which presents new challenges in use of these agents in the clinic .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Working in tandem with AK-A, the microtubule associated TPX2 (Targeting protein for Xklp2) protein plays vital role in spindle formation [, , ].
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Pre-metaphase, TPX2 assists with the nucleation of microtubules required for the mitotic spindle .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
During metaphase, TPX2 localizes and allosterically activates AK-A. The allosteric binding of TPX2, induces a conformational change that not only activates AK-A, but also prevents deactivation through phosphatase activity .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
TPX2 is also a substrate of AK-A. AK-A activated TPX2 has been shown to play a role in the maintenance of mitotic spindle length during mitosis .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Two potential strategies to cull CIP DLBCL cells are to promote mitotic catastrophe in proliferating polyploid cells, and/or disrupt polyploid cell formation, thus maintaining sensitivity to standard of care chemotherapy .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Hence the importance to establish the interrelationships of CIP cells and quiescence, senescence, and cell cycle dysregulation.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Thus, deciphering the mechanistic kinetics and dynamics of polyploidization, cell cycle dysregulation, and depolyploidization are key to identifying and designing novel therapeutic strategies to disrupt drug resistant CIP cells.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
We surmise that CIP cells acquire novel inheritable genetic changes to generate drug resistance progeny (2n+) via depolyploidation post-therapy.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Extensive analysis of the DNA content of two Myc-Bcl2 DLBCL cell lines demonstrated that alisertib therapy dramatically promotes polyploidization, as previously reported by others in DLBCL .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
By conducting cell cycle analysis using fluorescent reporter, we can start to explain this phenotype and address it using a TPX2 site inhibitor Here we describe the use of Aurkin A, an AK-A TPX2 site inhibitor, which prevents TPX2-AK-A interaction.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Combination of Aurkin A plus alisertib disrupts alisertib induced polyploidy in a dose-dependent manner with associated increased apoptosis.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Using the fluorescence ubiquitination-based cell cycle indicator (FUCCI) system we demonstrate by live cell imaging DLBCL cells treated with alisertib accumulate in S-G2 phase .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Importantly, a sub-population does not synchronize in S, but rather undergoes multiple division cycles via endomitosis compared to control.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Similarly, this endomitotic DNA replication was not observed with Aurkin A (TPX2/AK-A inhibitor) or alisertib plus Aurkin A combination DLBCL cells.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Thus, total AK-A inhibition was able to limit cell cycle slippage and is synthetic lethal to CIP cells.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 (DSMZ; ACC 633) and VAL (DSMZ; ACC 586)) cells were grown in RPMI-1640 media supplemented with 10% Fetal Bovine Serum.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
HeLa (ATCC; CCL-2), MDA MB-231 (ATCC; HTB-26), U-2 OS (ATCC; HTB-96), Granta-519 (DSMZ; ACC 342), and MIA PaCa-2 (ATCC; CRL-1420) cells were grown in DMEM supplemented with 10% Fetal Bovine Serum.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
All cells have been authenticated and are mycoplasma negative.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were grown in a 5% CO2 incubator at 37°C.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
All drug treatments were administered in a final volume of 1% DMSO.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Drug treatments for flow cytometry and immunostaining were performed over a period of five days, with a readministration of the corresponding drug and fresh media at day three.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 cells treated for five days with 1uM alisertib or DMSO, were fixed onto microscope slides using 5% paraformaldehyde solution, and treated with a nuclear extraction buffer (10mM PIPES,100mM NaCl, 300mM sucrose, 3mM MgCl2,1mM EGTA, 0.5% tritionx-100) to remove the cell membrane and cytoplasm.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were then blocked for one hour using 4% Bovine Serum Albumin solution.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
After blocking slides were washed three times with TBS-T and then stained with DAPI and rabbit CENP-B (Cell signaling Technology).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were washed, counterstained with anti-rabbit FITC conjugated antibody.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Imaging was performed using a confocal microscope at 40x magnification.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Drug treated U2932 and VAL, cells were fixed for 24-hours at 4°C in a 70% methanol solution.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were stained for one hour using a propidium iodide solution (100 mg/mL RNase, 150 mg/mL propidium iodide, and 5 mM EDTA-pH 8.0) and analyzed via flow cytometry for 100,000 events.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Results were quantified and plotted using FlowJo.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 cells were drug treated over a period of five days washed with fresh media and allowed to grow for an additional four days without drug.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
On days five and nine cells were collected and stained with PI and FITC Annexin V apoptosis kit (Biolegend).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were analyzed via flow cytometry for 100,000 events within an hour of initial staining and cell populations were quantified in FlowJo.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 and VAL cells were then assessed for cell proliferation via CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega, G5421).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
96-well plates were seeded with 10,000 cells per well in 100uL aliquots of colorless RPM 1640 media with 10% FBS.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Drugs were added to each well with a 1% final volume of DMSO.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Each condition was tested in four replicates.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Plates were incubated in a 5% CO2 incubator at 37°C for four days.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
20µL of via CellTiter 96 AQueous One Solution Reagent was added to each well and was incubated for an additional four hours.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
The absorbance of each plate was recorded via an absorbance plate reader at 490nm.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cell proliferation assay results were graphed using GraphPad prism, and the IC50, IC75, and IC90 of alisertib with and without 100µM Aurkin A was identified.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Using the Loewe additivity method, the combination index was calculated to determine if the addition of 100µM Aurkin A was synergistic at each inhibitory concentration .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
An isobologram of the combination indexes was created using GraphPad Prism.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were established like previously described .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Briefly, plasmids containing the fluorescent markers Geminin-clover (green) and cdt1-mKO (red), were used to transfect 293FT cells and generate lentiviral particles.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Lentiviruses were used to infect U2932 cells and generate stable cell lines upon Zeocin selection.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
The FUCCI U2932 cell line was then FACS sorted for the total population of dual green and red (yellow) fluorescence.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were then allowed to recover and grow for one week and then were subjected to a second FACS sorting, in which the top 10% of green cells were sorted and expanded.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
This homogenous population of FUCCI cells was synchronized using a double thymidine block .
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
After block release cells were drug treated and live imaging was performed using an Incucyte (Essen) imaging system.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Images were taken every 30 minutes and used for quantification and analysis.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Quantification of total green and red fluorescence (A.U) was plotted over time in GraphPad.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
A representative example of images was used for figures (as detailed in figure legends) while full time courses are shown as supplemental movies.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
SCID mice (Jackson laboratory) were injected with 1 × 10 VAL cells in sterile saline subcutaneously in the left flank.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Mice were pair matched based on tumor volume three weeks post injection and grouped into an alisertib 20mg/kg, Aurkin A 30mg/kg, Alisertib 20mg/kg + Aurkin A 30mg/kg combination, and vehicle control (DMSO volume match), each containing six mice.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Two mice in the Aurkin A and one mouse in the Alisertib treatment groups did not exhibit tumor growth post cell injection and were excluded.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Mice were given the corresponding treatment once a day for five days a week.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
After two weeks, treatment was then withheld for a week, followed by another five-day-a-week treatment cycle.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Paired T tests were used to evaluate the tumor volume differences between the groups.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
The tumors were then dissected and formalin-fixed paraffin-embedded.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Slides were stained with hematoxylin and eosin by the Tissue Acquisition and Cellular/Molecular Analysis core facility at the University of Arizona.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
All procedures were completed in accordance with the University of Arizona Institutional Animal Care and Use Committee (IACUC) protocol 07-029.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Mouse tumor slides were scanned at 20x magnification by The University of Texas Health Science Center at Houston Histology Core Facility.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Tumor slides were analyzed using the QuPath Bioimaging Analysis software (V0.4.3).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Deconvoluted H&E scanned slides were first evaluated for cell detection using the image optical density sum.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were then classified as polyploid or normalploid based nucleus to cell area ratio.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cell ploidy classification was repeated in three random areas of each slide for at least 10,000 cells per field.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Results were graphed via GraphPad Prism.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
To determine the effectiveness of inhibition of TPX2 binding to AK-A to prevent polyploidy, the effectiveness of alisertib to induce polyploidy was first established.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 cells (Myc/Bcl2 double expresser, DLBCL) were treated with alisertib at increasing concentrations from 1nM to 1M for five days.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
These cells were harvested, stained using a 10% PI solution, and analyzed via flow cytometry for DNA content to establish cellular ploidy.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Flow cytometry showed an increased level of 4n and 8n aneuploid cells with increasing alisertib concentration (Fig. 1a and Sup Fig. 1).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
The highest-level polyploidy after five days of alisertib treatment was observed at 1µM, with 4n and 8n aneuploid cells making up 40% and 25% of the cell population respectively (Fig. 1b).Fig.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
1Alisertib induced polyploidization in Diffuse Large B Cell Lymphoma.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 cells treated with DMSO, 50nM, 100nM, 500nM, or 1000nM alisertib for five days were fixed in 70% methanol solution, stained with PI, and analyzed via flow cytometry (A, B).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 cells were treated with DMSO (C) or 1µM Alisertib (D).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were treated stained with CENP-B and DAPI and imaged at 40x magnification via confocal microscopy.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Fig 1 Alisertib induced polyploidization in Diffuse Large B Cell Lymphoma.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 cells treated with DMSO, 50nM, 100nM, 500nM, or 1000nM alisertib for five days were fixed in 70% methanol solution, stained with PI, and analyzed via flow cytometry (A, B).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
U2932 cells were treated with DMSO (C) or 1µM Alisertib (D).
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells were treated stained with CENP-B and DAPI and imaged at 40x magnification via confocal microscopy.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
To further investigate ploidy changes in alisertib treated U2932 cells, immunofluorescent (IF) confocal microscopy was utilized.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Cells treated for five-day with either 1mM alisertib or vehicle (DMSO) were collected and stained with both a CENP-B antibody and the nuclear stain DAPI.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
CENP-B, a highly conserved centromere protein, is commonly used to help with chromosome observations and numeration.
PMC11225860
Aurkin-A, a TPX2-aurora a small molecule inhibitor disrupts Alisertib-induced polyploidy in aggressive diffuse large B cell lymphoma
Microscopy showed a drastic increase in nucleus size between treatment and control, as 1µM alisertib treated nuclei were more than five times larger than DMSO (Fig. 1c and d).