Evaluating c-Myc gene regulation of PIM2 in Multiple Myeloma

Authors & Affiliations

Christopher Schorr [1,2]

[1] Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN

[2] Indiana University School of Medicine (IUSM), Indiana University, Indianapolis, IN

Abstract

Multiple Myeloma (MM) is a malignancy of long-lived plasma cells (PCs) that reside in the bone marrow (BM). MM burdens patients with life-altering disruptions in the production of normal blood cells, bone integrity, kidney function, and immune response (1). MM accounts for 1.8% of all cancers and 10-15% of all hematological cancers worldwide, second only to non-Hodgkin’s lymphoma, with 35,730 new cases (56%:46% M:F) and 12,590 deaths expected in 2023 (American Cancer Society) (2,3). Prior to 2000, the average 5-year MM survival rate did not exceed 35% but since the recent introduction of new therapies such as proteasome inhibitors, monoclonal antibodies, and chimeric antigen receptor (CAR). Nevertheless, MM remains an incurable disease for almost all patients, primarily due to treatment resistance which is linked to the upregulation of adaptive pro-survival mechanisms in MM cells. PIM (Proviral Integration site in Moloney Murine Leukemia Virus) proteins are a family of putative threonine/serine kinase oncoproteins that have been identified in cancer since 1986. In particular, PIM2 expression is most elevated in MM and is correlated with a poor prognosis and disease progression in MM (4). Using in-silico analysis, I have identified several MM-disease relevant putative transcription factors (TF) binding sties on the PIM2 promoter region – including c-MYC, MAX, STAT3, IKZF1, and IRF4 (Figure 1). c-Myc serves a “master regulator” of cellular metabolism and proliferation (5). Many cancers including MM are dependent on c-Myc activity for survival (6). We have previously shown that inhibiting c-Myc decreases PIM2 expression, however very little is known in the mechanism by which inhibiting c-Myc regulates PIM2 expression in MM patients. My hypothesis is that c-Myc binds to cis-regulatory elements (CRE) of the PIM2 promoter and leads constitutively elevated levels of PIM2 in MM cells. Using chromatin immunoprecipitation and dual luciferase reporter assays, I will test this hypothesis by determining the precise binding of c-Myc and its activity on PIM2 promoter gene regulation in MM cell lines (Figure 2). I am co-advised by Dr. Xiaoping Bao (researcher studying TF regulation in stem cell differentiation) and Dr. Kelvin Lee (MM oncologist and Director of the IU Simon Comprehensive Cancer Center).

Introduction

Motivation

Multiple myeloma (MM) remains an incurable hematologic cancer characterized by uncontrolled plasma cell proliferation in bone marrow, representing the second most common blood malignancy. Recent therapeutic progress has extended median survival to 6 years, however MM uniformly acquires lethal multi-drug resistance by upregulating pro-survival mechanisms including PIM2 signaling. We find PIM2 kinase is specifically overexpressed in advanced MM patients where it enables pathogenesis and therapeutic evasion by promoting cancer cell survival through diverse mechanisms regulating translation, metabolism, cell cycle, and bone microenvironment interactions. Yet how PIM2 blockade can prevent and overcome drug resistance to improve patient outcomes remains unknown.

Impact/Significance

Multiple myeloma continues to have high mortality rates, largely due to the development of drug resistance. The goal of this project is to elucidate the specific mechanisms by which multiple myeloma cells evade current standard therapies. Using state-of-the-art genomic and proteomic profiling, we will identify novel pathways and targets involved in therapeutic resistance. These findings will directly inform our subsequent aims to develop and validate next-generation treatments designed to override resistance mechanisms. By improving outcomes for this deadly blood cancer, our research has the potential to serve as a model for advancing new therapeutic approaches against other resistant malignancies. Given the pressing need for innovative treatments for these patient populations, our study addresses an issue of high significance in oncology and public health. The knowledge gained through our integrated approach combining functional genomics and drug testing provides a unique opportunity to make a substantial impact on this challenging disease.

Hypotheses/Aims

This project aims to definitively establish c-MYC as a direct regulator of aberrant PIM2 transcription by integrating bioinformatics, genomic editing, chromatin immunoprecipitation (ChIP), overexpression, and CRISPR knockout/interference approaches. Putative c-MYC response elements will be predicted computationally and functionally validated using luciferase reporter assays. c-MYC binding will be confirmed at endogenous regulatory regions by ChIP-qPCR. Necessity and sufficiency for governing PIM2 expression will be evaluated through modulation studies.

Materials/Methods

To validate findings, I will utilize a comprehensive collection of well-characterized human MM cell lines and primary MM cells obtained from patient samples. Through active research collaborations, our lab has access to extensive biobanks of viably frozen myeloma samples, including over 6,000 bone marrow and peripheral blood specimens curated in the IUSCCC Indiana Myeloma Registry and Roswell Park Comprehensive Cancer Center Hematologic Procurement Shared Resource. This large patient dataset representing all stages and genetic subtypes of myeloma will enable confirmation of cell line results in primary cells, as well as assessment of clinical correlates with expression and therapeutic relevance.

Study type

Experiment - A researcher randomly assigns treatments to study subjects, this includes field or lab experiments. This is also known as an intervention experiment and includes randomized controlled trials.

Existing data

Figure 1. Putative PIM2 Cis-regulatory elements (CRE). Histone marks shown determined by ChIP-seq in MM.1S cells (GSE151556). Binding of pertinent transcription factors (TFs) to PIM2 CRE defined by TF ChIP-seq Clusters from ENCODE3.

Figure 2. Preliminary ChIP data for c-MYC binding on PIM2 promoter. #1 through #7 represent consecutive ~1,000 bp regions on the PIM2 promoter from +4.6 kb to -2.6kb from PIM2 TSS. RPL30 represents positive ChIP control. Dark grey bars are negative IgG controls.

Data collection procedures

Luciferase Reporter Assays Putative c-MYC responsive promoter elements will be PCR amplified and cloned upstream of the luciferase gene in the pGL4 vector. Constructs will be sequence verified prior to transient transfection in triplicate into MM1.S and RPMI 8226 cells using lipofection. A Renilla plasmid will be co-transfected as an internal control. Luciferase activity will be measured 24 hours post-transfection using the Dual-Glo system (Promega) and normalized to Renilla values.

CRISPR Activation Screen A lentiviral dCas9-VP64 sgRNA library will be transduced into MM cells at an MOI of 0.3 to maintain ≥500 cells per sgRNA. Transduced pools will be selected with blasticidin then assayed for target gene expression by RT-qPCR. Two replicates per sgRNA will be evaluated.

ChIP-qPCR Chromatin immunoprecipitation will be performed using a c-MYC antibody or IgG control. Immunoprecipitated DNA will be quantified by qPCR using primers flanking putative c-MYC binding sites within target gene regulatory regions. Fold enrichment over background will be calculated using the ΔΔCt method relative to input DNA.

Overexpression Studies c-MYC will be overexpressed by transient transfection of an expression vector containing cDNA under control of a constitutive promoter. Empty vector will serve as a negative control. mRNA and protein levels will be evaluated by RT-qPCR and Western blotting, respectively, 48 hours post-transfection.Sample size

To validate c-MYC responsive elements, we will perform luciferase reporter assays by transiently transfecting constructs in triplicate into MM cell lines, providing 90% power to detect a ≥1.5 fold change in promoter activity (α=0.05) based on preliminary data. The CRISPR activation screen utilizing dCas9-VP64 and an sgRNA library will transduce cells at an MOI of 0.3 while maintaining ≥500 cells per sgRNA during selection, in line with guidelines for reasonable sensitivity in detecting activating sgRNAs. Each sgRNA will be evaluated in duplicate. For ChIP-qPCR, we will use n=3 biological replicates per condition, providing 80% power to detect a ≥2-fold enrichment over background (α=0.05) based on preliminary ChIP and published recommendations. Finally, overexpression experiments will utilize n=4 biological replicates to achieve 90% power for detecting a ≥1.7-fold change in mRNA or protein levels (α=0.05). These sample sizes are based on preliminary data and published methodological guidelines to ensure adequate power for interpreting results.

Budget

Costs

Item and description	Quantity and Unit	Unit Price	Total
SimpleChIP® Plus Sonication Chromatin IP Kit (Cell Signaling #56383)	(4 kits)	$530.00	$2,120.00
c-Myc Antibody (Proteintech 10828-1-AP)	(2 vials – each 150 μl)	$389.00	$778.00
pGL4-Basic Vector (Promega)	1 plasmid stock	$340.00	$340.00
pRL-CMV (Promega)	1 plasmid stock	$340.00	$340.00
Q5 Site-Directed Mutagenesis Kit (New England Biolabs)	(2 kit - each 10 reactions)	$224.00	$448.00
Core Facilities	N/A	N/A	Variable (estimate $974.00)

Note: all necessary cell culture equipment and laboratory space is to be provided by Dr. Xiaoping Bao and Dr. Kelvin Lee.

References

Cowan AJ, Green DJ, Kwok M, Lee S, Coffey DG, Holmberg LA, et al. Diagnosis and Management of Multiple Myeloma: A Review. JAMA. 2022 Feb 1;327(5):464–77.
Elbezanti WO, Challagundla KB, Jonnalagadda SC, Budak-Alpdogan T, Pandey MK. Past, Present, and a Glance into the Future of Multiple Myeloma Treatment. Pharmaceuticals. 2023 Mar 8;16(3):415.
American Cancer Society | Cancer Facts & Statistics [Internet]. [cited 2023 May 8]. American Cancer Society | Cancer Facts & Statistics. Available from: http://cancerstatisticscenter.cancer.org/
Liu Z, Han M, Ding K, Fu R. The role of Pim kinase in immunomodulation. Am J Cancer Res. 2020 Dec 1;10(12):4085–97.
Miller DM, Thomas SD, Islam A, Muench D, Sedoris K. c-Myc and Cancer Metabolism. Clin Cancer Res Off J Am Assoc Cancer Res. 2012 Oct 15;18(20):5546–53.
Holien T, Våtsveen TK, Hella H, Waage A, Sundan A. Addiction to c-MYC in multiple myeloma. Blood. 2012 Sep 20;120(12):2450–3.
Mikkers H, Nawijn M, Allen J, Brouwers C, Verhoeven E, Jonkers J, et al. Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors. Mol Cell Biol. 2004 Jul;24(13):6104–15.
Li S, Vallet S, Sacco A, Roccaro A, Lentzsch S, Podar K. Targeting transcription factors in multiple myeloma: evolving therapeutic strategies. Expert Opin Investig Drugs. 2019 May;28(5):445–62.

Funding

Funding

Evaluating c-Myc gene regulation of PIM2 in Multiple Myeloma

Funding Progress

Nonprofit Recipient

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