Intratumoral heterogeneity in cellular response to p53 and ISR activation

About this position

• Doctoral study program: Biomedical Sciences
• Form: doctoral (present)
• Department: Department of Biology
• Supervisor: Zdenek Andrysik, Ph.D.

Ph.D. position:

Intratumoral heterogeneity in cellular response to p53 and ISR activation

Annotation:

The most frequently mutated tumor suppressor gene in human cancers is TP53, which encodes

transcription factor p53. Induced by various types of cellular stress, p53 transactivates a broad range of

genes involved in numerous anti-cancer programs including cell cycle arrest, DNA repair, senescence,

and apoptosis. However, targeted induction of p53 by small molecule inhibitors of a key upstream

negative regulator MDM2 leads in most cancer types to a reversible proliferation block, while the

therapeutically desirable elimination of cancer cells by apoptosis is limited at best. Recently, we identified

a novel regulatory mechanism within the p53 network (Andrysik et al., Nature Communications, 2022)

responsible for restraining the apoptotic response upon p53 activation. In this scenario, p53 activity

maintains eukaryotic translation initiation factor eIF2a dephosphorylated and fully functional. Severing

such negative feedback loop pharmacologically by eIF2a inhibitors leads to rapid onset of apoptosis in

cancer cells in vitro and stalled tumor growth and animal survival in vivo.

The key shortcoming of the combined therapy resides in insufficient apoptotic response in treated

tumors. Preliminary immunohistological (IHC) analysis of colorectal carcinoma (CRC) xenograft tumors

revealed variable induction of p53 and ISR in individual cells suggesting existence of treatment-resistant

subpopulations. We hypothesize that tumor cell subpopulations unsusceptible to targeted

pharmacological activation of p53 and ISR pathways bear distinct characteristics which can be

explored therapeutically. Therefore, we propose two complementary strategies to test our hypothesis:

First, we will characterize activity of both targeted pathways in individual tumor cells by

transcriptome analysis. Numerous factors impinge drug efficiency in vivo and no available models

recapitulate complexity of tumors developing at specific organismal locations. We hypothesize that

tumor subpopulations resistant to MDM2 and eIF2a inhibitors may be targeted pharmacologically

to improve the overall response to combination treatment. Therefore, we will perform a single-cell

RNA-seq (scRNA-seq) in orthotopic CRC tumors following combined treatment with MDM2 and eIF2a

inhibitors. Next, we will score p53 and ISR activity in individual tumor cell clusters and characterize in

detail cellular processes and pathway activities in both responding and non-responding subpopulations

using bioinformatical tools for transcriptome interpretation. Finally, we will propose and test strategies

for sensitizing resistant populations both in vitro and in vivo.

Second, we will map p53 and ISR responses over the treatment course. It is unclear whether

the key resistance mechanisms are either innate to the tumor or develop as a response to the treatment.

We hypothesize that tumor subpopulations resistant to MDM2/eIF2a inhibitors will bear distinct

characteristics in short-time and long-time treatments. To elucidate changes in tumor subpopulations

and to define dynamics of resistance development, we will perform both IHC and spatial transcriptomics analyses of orthotopic CRC tumors treated with a combination of MDM2 and eIF2a inhibitors in a single dose and over a two to four week-long treatment regimen. Next, we will test strategies preventing resistance development and increase efficiency of the combined treatment.

Funding of the research:

Primary funding for the proposed work is secured by AZV award NW25-03-00134 to the supervisor.

Information on funding PGS positions:

The program requires that all PhD students have some means of financial support of min. 25 000 CZK per month. This is often a combination of various sources (grants, scholarship etc.)

Requirements for the student according to the Doctoral Board:

The student's minimum publication activity within the course of study is one first-author publication with an IF value above the median in the field or 2 first-author publications in journals with an IF value in the 3rd quartile in the field (Q3). A condition for successful completion of the studies is also a foreign internship of at least 1 month, which is an inseparable part of the studies. As part of their studies, students will also participate in the teaching.

Additional reading:

https://pmc.ncbi.nlm.nih.gov/articles/PMC9715646/