Oncology

• Chronic myeloid leukemia (CML) is a type of leukemia that starts in the bone marrow and then spreads to the blood.
• Two types of stem cells:

  • Embryonic: stem cells can self-renew and give rise to all types of mature cells
  • Adult: stem cells can self-renew and give rise to mature cells of the specific organ or tissue
• Main features of stem cells:

  • Life-long support of mature cells in a quiescent state to avoid differentiation and exhaustion of the stem cell pool
  • Self-renewal
  • Proliferation and differentiation into several progenitor cells and further on to mature cells
• Cancer Stem Cells:

  • Rare cells within tumors with the ability to self-renew and give rise to the phenotypically diverse tumor cell population to drive tumorigenesis
• Stem cell differentiation hierarchy:

  • Increased plasticity may be present within different cancer cell populations, enabling bidirectional interconvertibility between CSCs and non-CSCs
• Origin of the Theory of Cancer Stem Cells:

  • Only a small subset of cancer cells is able of extensive proliferation
  • A large number of cells are required to grow tumors in xenograft models
  • CSCs may have different sensitivities to radiation or chemotherapy
• Tumor ecosystem:

  • Tumor is a complex ecosystem containing tumor cells, various infiltrating endothelial, hematopoietic, stromal, etc. cell types
  • The frequency of CSCs in a tumor is highly variable
  • CSCs can be prospectively identified
  • CSCs may have different sensitivities to radiation or chemotherapy
• Regulation of stem cells:

  • Signaling pathways and transcription factors regulating CSCs:
  • Wnt signaling is one of the main stem cell regulating pathways
  • Epithelial-to-mesenchymal transition (EMT) in metastatic CSCs
  • Oncogenic Reprogramming Induced by Mutated Epigenetic Regulators
• Alternative techniques for CSCs:

  • Zebrafish transgenic or xenotransplantation models
  • Patients-derived induced pluripotent stem cells (iPSCs)
  • Organoids
  • CRISPR/Cas-, ZNF-, or TALEN-mediated gene editing by introducing LSCs-associated gene mutations or correction of mutations
  • NGS-based multi-OMICs approaches including single-cell RNA-Seq, DNA-seq, ATAC-Seq, ChIP-Seq, CUT&RUN, Methylomics, Imaging, etc.
• Therapeutic strategies to target CSCs:

  • Tumors are heterogeneous ecosystems with some cells having stem cell-like features, so-called cancer stem cells (CSCs)
  • Different levels of CSCs regulation
  • Stem cell markers including CSCs are often shared between stem cells of different tissues or organs
• Models of Carcinogenesis:

  • Stochastic model: All isolated tumor cells have the capacity to differentiate indefinitely and form new tumors
  • Cancer stem cell model: Only cancer stem cells have the ability to form tumors
• CRISPR-CAS9 Gene Editing:

  • Application of CRISPR Technology
  • Editing of individual cells
  • Combinatorial editing: Sequential editing and simultaneous editing
  • CRISPR pooled screening
• Cell lines:

  • Pros: indefinite source of biological material for experimental purposes, easy to manipulate, standardized protocols
  • Cons: high risk of contamination, no pre-cancer lines, complex and lengthy establishment process
• Mice models:

  • NOD-Prkdcscid-Il2rgnull (NSG)
  • NOD-Rag1null-Il2rgnull (NRG)
  • CB17/Icr-Prkdcscid (SCID)
  • BALB/c Nude Mouse
• Xenografting of Human Cancer Cell Lines and PDXs into Zebrafish Larvae:

  • Disease Modeling in Zebrafish Pros and Cons
• iPSCs and Disease Modeling:

  • Isolation of somatic cells
  • Reprogramming by TFs
  • Gene editing
• Datasets generated from Cancer cell lines:

  • Cancer Cell line Encyclopedia
  • Genomics of Drug Sensitivity in Cancer
  • MD Anderson Cell Lines Project
  • Depmap portal (ProjectAchilles)
  • Cell Model Passports
• Reprogramming by transcription factors (TFs) Oct4, Sox2, Klf4, and cMyc
• Advantages of iPSC-Based Disease Modeling System:

  • Differentiation to virtually any cell type
  • Eliminates ethical issues
  • Consistent and well-controlled phenotypes for disease modeling
  • Continuous cell supply
  • Possible preservation
  • Availability and accessibility of source cells
  • Personalization of treatment and diagnostics
• Limitations of iPSC-Based Disease Modeling System:

  • Candidate gene essential for iPSCs may not tolerate introduction of loss-of-function mutations
  • Reprogramming adult cells into iPSCs is relatively inefficient and time-consuming
  • Limited potential for high-throughput applications due to high costs, timeline, and expertise required
  • Guiding iPSCs to differentiate into specific cancerous cells is technically challenging and inefficient
  • iPSC line-to-line variability in phenotypic readouts
  • iPSC-derived cells may have impaired ability for terminal differentiation
• • Lack of complex microenvironment in iPSC system
• Organoids:

  • 3D system imitating the architecture and functionality of native organs
  • Contain self-renewing stem cell populations that can differentiate into specific cell types in organ tissues
  • Richer cell composition and physiological function compared to traditional 2D cell culture
  • Widespread use in biological function research, artificial organ development, disease modeling, and drug screening
• Organ-on-a-Chip (OOC):

  • Microfluidic devices replicating the structure and function of organs or tissues on a chip
  • Multiple applications including static cultures of multiple cell types, perfused cultures of a single cell type, and perfused cultures of multiple cell types
• Components of the tumor microenvironment:

  • Structural components:
  • Extracellular matrix (ECM) composed of filament- and network-forming proteins and glycans
  • Blood and lymphatic vessels providing nutrients, oxygen, and pathways for metastatic cells
  • Cellular components:
  • Endothelial cells, stromal cells, and immune cells (T cells, macrophages, dendritic cells, natural killer cells, neutrophils, B cells)
  • Soluble factors:
  • Cytokines, chemokines, growth factors, and metabolites
• Functions of the extracellular matrix in the tumor microenvironment:

  • Drug diffusion barrier
  • Escaping immune response
  • Promoting invasion and metastasis
  • Limiting tumor cell invasion and mediating DNA damage
  • Mediating survival signals
• Cytokines, chemokines, and growth factors main effects in the tumor microenvironment:

  • Mediate cell survival or apoptosis
  • Mediate cell recruitment
  • Induce cell differentiation
  • Modulate the immune response
• Immunoediting of cancer cells and their microenvironment:

  • Cold Tumor (Immune escape) characterized by the exclusion of CD8 T and NK cells, presence of immunosuppressive cells, and poor prognosis
  • Hot Tumor (immune equilibrium) infiltrated by CD8 T and NK cells, absence of immunosuppressive cells, and better prognosis for immunotherapies
• Diversity of tissue niches and microenvironments in metastatic cancer:

  • Clinical challenge due to heterogeneity between microenvironments of metastatic sites
  • Coexistence of diverse immune evasion and therapy resistance mechanisms within one cancer patient
  • Need for synergistic combination therapies to target multiple resistance and therapy evasion mechanisms
• Strategies to co-target the microenvironment:

  • Vessel normalization (Anti-VEGFα)
  • Immune checkpoint inhibition
  • Targeting fibroblasts
  • Directly targeting ECM (e.g., targeting proteases in liposomes to digest ECM)
• 8 out of 10 of the hallmarks of cancer are regulated by the microenvironment
• RNAs in nature have various functions, including:

  • Splicing (self splicing introns)
  • Enzyme (ribozymes, riboswitches)
  • Protein Synthesis (rRNA, tRNA)
  • Inhibition (miRNA, siRNA, piRNA, CRISPR)
  • Protein coding (mRNA)
  • DNA Template (telomeres)
  • Chromosome inactivation (XIST in women silencing one X chromosome)
  • Transcriptional regulation (lncRNA)
• Therapy implications of RNA include:

  • Splicing → SSO (Spinraza)
  • Enzyme → gene therapy regulation
  • Protein synthesis → premature stop codon readthrough
  • Inhibition → siRNA (Onpattro)
  • Protein coding → Covid vaccines
  • DNA template → Gapmer - ASO (Kynamro)
  • Chromosome inactivation → Down-syndrome
• RNA therapeutic design must:

  • Be recognized and bound by proteins necessary for the function
  • Be metabolically stable for an appropriate amount of time
  • Have appropriate immunogenicity
  • Be able to get delivered to target tissues and cells
• mRNA Applications include:

  • Vaccine for infectious disease or cancer
  • Protein replacement therapy
  • Temporary CAR expression in endogenous T cells
  • mRNA based CRISPR for enhanced safety
  • mRNA based conditioning before HSCT (pro-apoptotic mRNA)
• mRNA modifications in approved RNA therapies are limited to modifications found in nature
• mRNA Toxicities can include:

  • Immunogenicity: sequence-specific/structure-specific
  • Lipid nanoparticle (PEG, cationic lipids)
  • Modified bases recycling
• Aptamers are short sequences of artificial DNA, RNA, XNA, or peptide that bind a specific target molecule or family of target molecules. Examples include:

  • Pegaptanib (Macugen) approved in 2004 for VEGF in Age-related macular degeneration
• siRNA applications include:

  • Targeting any mRNA or lncRNA
  • Specific to splice variants
  • Differentiating alleles based on SNPs
  • Long-lasting effect
  • Effect sensitive to proliferation rate
• ASO (antisensoligos) applications include:

  • Pre-mRNA degradation
  • Splice switching
  • Translation modulation
  • N=1 medicines
  • RNA editing
• RNA versus gene therapies:

  • Temporary effect
  • Can be turned off anytime
  • Smaller for easier delivery
  • Chemical entity, not biological entity
  • No genotoxicity
  • "Library-effect" for individualized therapy as N=1 or N=few medicine