Study of Cancer Stemness in Contributing Intra-tumoral Heterogeneity in ALK-positive Anaplastic Large Cell Lymphoma
Creators: Shang, Chuquan
Publisher: name: University of Alberta Library
Year: 2024

<p>Anaplastic large cell lymphoma (ALCL) expressing anaplastic lymphoma kinase (ALK+ALCL) is a distinct subtype of non-Hodgkin lymphoma characterized by the presence of the ALK protein due to the reciprocal t(2;5)(p23;q35) chromosomal translocation. It is a specific type of aggressive non-Hodgkin lymphoma (NHL) of the T-cell lineage. ALK+ALCL is typically associated with a more favorable prognosis compared to ALK-negative (ALK-) ALCL, mostly due to the presence of targeted treatments that precisely target the ALK protein. The current first-line treatment typically involves the utilize of traditional standard chemotherapy in addition of FDA-approved ALK inhibitors. These medications have further demonstrated enhanced safety, selectivity, and potency. Although targeted therapy has greatly enhanced the prognosis for patients with ALK+ALCL, the occurrence of relapse and refractory still pose considerable obstacles. Resistance to ALK inhibitors is a contributing factor to the recurrence of the disease. Current research is focused on developing new and more effective treatments and drugs that target ALK inhibitors to address the difficulties faced and enhance the results for patients. One of the well-accepted hypothetical concepts is that cancer stemness, which was driven by a small set of population equipped with stem cell features (known as Cancer Stem Cells, CSCs), is believed to play critical roles in cancer relapse and the development of refractory diseases, thus challenging the long-term efficacy of therapies through intra-tumoral heterogeneity. <br>
The key aim of this study was to study the potential mechanisms underlying the hypothesis of cancer stemness in contributing to intra-tumoral heterogeneity in ALK+ALCL. The Lai Lab has previously applied a commercially available Sox2 regulatory region 2 (SRR2) reporter to identify a small subset of Reporter Responsive (RR) cells being more stem-like in terms of being more tumorigenic and chemoresistance when compared to the bulk Reporter Unresponsive (RU) cells. Additionally, previous results have shown that down-regulation of Myc in RR cells can phenotypically convert them to RU cells; opposite effects were observed with Myc protein overexpression in RU cells. However, the detailed mechanisms of distinguishable Myc protein expression in contributing to this intra-tumoral heterogeneity are still unclear.<br>
In this study, I first verified the chemoresistance in the stem-like subset RR cells in responding to the targeted therapeutic drug Crizotinib. Furthermore, by employing a dual fluorescence tagged exogenous LC3 plasmids, I found this resistance was due to the cytoprotective autophagy mediated by the higher Myc protein expression in RR cells as they can be sensitized to Crizotinib by Myc downregulation. Next, to examine the detailed mechanism of how Myc stayed at a relatively higher expression level, I studied the NPM1-mediated Myc proteasomal degradation (PD-Myc) pathway as there was no difference between RU and RR cells in terms of Myc mRNA expression. While both NPM1 and NPM-ALK contain the oligomerization domain at the C-terminal region, the formation of NPM1 homodimer, NPM-ALK homodimer, and NPM-ALK:NPM1 heterodimer were observed. By employing size exclusion chromatography and immunoprecipitations, I detected the different complexity and distribution of nuclear NPM-ALK:NPM1 heterodimers between RU and RR cells. More specifically, more nuclear heterodimers trended to form larger complexes in RR cells, sequestering the E3 ubiquitin ligase Fbw7γ, which is responsible for the ubiquitinated PD-Myc within the nucleus, thus resulting in the deficient PD-MYC and maintaining a relatively higher Myc protein expression level in RR cells. Lastly, while nuclear function of NPM-ALK is bared studied, given the fact that NPM1 is a multifunctional protein, it’s reasonable to hypothesize that the normal function of NPM1 in the nucleus could be disrupted when forming dimerization with nuclear NPM-ALK. By using label-free mass spectrometry and pathway enrichment analysis, this turned out to be the case. Moreover, the application of immunofluorescence and confocal microscopy allowed me to further generate the hypothetical model that the nuclear NPM-ALK could induce intrinsic nucleolar stress responses as the survival mechanisms in RR cells, potentially contributing to the cytoprotective autophagy and inefficient PD-Myc.<br>
Overall, this study provided a novel direction of how cancer stemness is regulated in ALK+ALCL and set up an integrated pipeline and model to study the potential mechanism of cancer stem-like cell-regulated chemoresistance and intra-tumoral heterogeneity. While directly targeting Myc is still full of challenges at the clinical research level, this study also provided a potential new solution to indirectly regulate Myc and Myc-dependent chemoresistance.</p>