Ongoing projects
“Establishing the Importance of CTLH Proteins in Non-Small Cell Lung Cancer”
(Pelotonia Idea Award).
ABSTRACT
The ablation of RanBP9 (Ran Binding Protein 9) renders Non-Small Cell Lung Cancer (NSCLC) cells sensitive to DNA damaging agents such as ionizing radiation, cisplatin, ATR-, and PARP-inhibitors. Most importantly, the levels of RanBP9 protein assessed by immunohistochemistry inversely correlate with response to treatment with platinum-based drugs (Palmieri et al., 2016; Tessari et al., 2018; Tessari et al., 2020).
RanBP9 exerts its functions by modulating the stability of other proteins through a largely uncharacterized but evolutionary conserved multi-subunit E3 ligase called the CTLH (C-Terminal to LisH) complex. This structure includes 10 members. RanBP9 with Gid8 (Glucose-Induced degradation Deficient 8) provides a necessary building block on which the rest of the structure is aggregated. Maea (Macrophage Erythroblast Attacher E3 ligase) and Rmnd5 (Required in Meiotic Division 5) together provide the ligase enzymatic activity bearing each half of a RING domain. The role of a paralog of RanBP9, part of the complex and named RanBP10, has never been investigated.
The complex as a whole appears to be over-expressed in the vast majority of NSCLC patients (www.cbioportal.org). Recent evidence shows that the CTLH complex regulates WNT and AMPK signaling. All considered, the ablation of this complex could represent a formidable way to weaken NSCLC cells defenses against therapeutic strategies currently used, including immune therapies.
Based on evidence from S. cerevisiae, we hypothesized that RanBP10 works in concert with RanBP9 within the complex, which would be structurally and functionally destroyed when they are both absent. Therefore, the combined ablation of RanBP9 and RanBP10 would result in more severe vulnerabilities to DNA damage than those we observed upon deletion of RanBP9 alone. However, it is important to fully understand the relevance and the role of RanBP10 in NSCLC, which have never been investigated. We want to investigate if RanBP10 is highly expressed in NSCLC and whether its levels are tightly regulated in concert with RanBP9. In addition, we aim to test whether RanBP10 has a role similar to RanBP9 in responding to DNA damage by presenting proteins to be ubiquitinated to the CTLH complex.
The specific aims of our proposal are:
AIM1: Test whether the combined deletion of RanBP9 and RanBP10 causes increased and/or broader vulnerability to drug treatment.
AIM2: Ascertain the levels of both RanBP9 and RanBP10 protein expression in human NSCLC.
AIM3: Identify proteins bound by Scorpins following genotoxic stress.
The impact of this study will be high. While it is undeniable that targeted- and immune therapy have made significant progress in the treatment and management of NSCLC, it is also true that not always these therapies can be used and in most cases, they are not curative but have only temporary effects. Therefore, seeking new therapeutic avenues and potential targets is still an incredibly valuable goal.
This study will provide support to the idea of targeting RanBP9 and RanBP10 together as a potential therapeutic strategy in NSCLC. However, this proposal has also the merit and novelty of elucidating the molecular mechanisms in which an uncharacterized E3 ligase participates in responding to DNA damage.
“Turning RanBP9 on NSCLC”
(RO3 CA259389)
SUMMARY
Remarkable advancements have been recently achieved in the treatment of Non-Small Cell Lung Cancer (NSCLC). Targeted- and immune-therapy have produced spectacular patient remissions unimaginable only 5 years ago for this disease that is the number one cause of cancer related deaths in the developed world. Unfortunately, most patients do not achieve a durable response and are not cured. Therefore, there is still an urgent need for new therapeutic targets and strategies to treat this devastating malignancy.
The protein RanBP9 could be an attractive target for the treatment of NSCLC. RanBP9 protects NSCLC cells from stress induced by DNA damaging agents such as platinum based-drugs. The higher its levels, the worse the NSCLC patient response to platinum-based drugs. This scaffold protein is significantly over-expressed in NSCLC cells when compared to the normal counterpart, which is expected on the basis of its anti-stress protective functions. Thus, a therapeutic window might be open to target RanBP9 without damaging normal cells. On the other hand, like numerous other potential targets for cancer therapy, RanBP9 is widely expressed. Relatively high expression of RanBP9 is found in the brain and in the gonads, for example. Therefore, before taking the idea of inactivating RanBP9 to the clinics, careful work needs to be done to better understand how RanBP9 protects cells from DNA damage. Other than in cancer cells, it is particularly important to establish how this proteins works also in tumor associated macrophages (TAMs), which have been shown to promote tumorigenesis and resistance to therapy. To achieve these goals, whole organism modeling is necessary.
This proposal seeks to study by advanced proteomics the changes of the molecular interactions that RanBP9 shows in both normal and cancer cells when treated with cisplatin. To distinguish the interactions of RanBP9 in cancer cells from those in the rest of the tumor microenvironment, we will use a new mouse line called RanBP9-TURN in which RanBP9-HA is turned off and RanBP9-V5 is turned on in cells where Cre is active.
In the first specific aim (Aim 1) of this proposal, we will study the interactions of RanBP9 in type II pneumocytes and in alveolar macrophages in a mouse model in which RanBP9 tag is switched from HA to V5 upon Cre-recombination. This will establish the changes of the RanBP9-interactome following treatment with cisplatin in normal alveolar type II cells and alveolar macrophages. In the second aim (Aim 2), we will study the dynamics of RanBP9 protein interactions in NSCLC cells and TAMs upon DNA damage. This will reveal the RanBP9-interactome changes in cancer cells and TAMs following DNA damage by cisplatin.
When accomplished, this study will add support to the pursue of mechanistically-designed therapies to treat NSCLC based on the targeting of RanBP9. It will also open new lines of investigation into unknown mechanisms of the cellular response to DNA damage. Finally, the RanBP9-TURN will be a prototype in the field of mouse modeling for investigations into the tumor microenvironment.