Biochemical Foundations of Pharmaceutical Biotechnology PDF

Biochemical foundations of pharmaceutical biotechnology Oncogenes and their inhibition in managing cancer Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected] O...

Biochemical foundations of pharmaceutical biotechnology Oncogenes and their inhibition in managing cancer Audrey Minden, Ph.D. Department of Chemical Biology Ernest Mario School of Pharmacy Susan Lehman Cullman Laboratory for Cancer Research, Room 205 (848) 445-5766 [email protected] Oncogenes: Outline of topics covered: I. Overview of oncogenes A. Oncogenes compared with tumor suppressor genes B. Basic terminology used to discuss oncogenes C. Activation of oncogenes II. Viruses and early oncogene studies, including Src III. Identification of new oncogenes IV. Ras oncogene A. Normal vs oncogenic Ras B. Human cancers with Ras mutations V. Myc oncogene VI. Receptor tyrosine kinases that are oncogenes A. EGFVIII and drugs to target it in cancer B. Her2/Neu and breast cancer, and relevant drugs C. FGF receptor and bile cancer, and relevant drugs VII. Oncogenic Raf, and drugs designed to target it VIII. Activated PI3Kinase (PI3K) in cancer A. Mechanism of PI3K activation B. Drugs designed to target activated PI3K IX. CDK4 in cancer, and drugs designed to target CDK4 2 Oncogenes Vs. Tumor Suppressor Genes 3 Oncogenes compared with tumor suppressor genes Oncogene encodes an oncoprotein Tumor suppressor gene encodes a tumor suppressor protein (figure from Alberts) 4 Oncogenes, Proto-oncogenes, Tumor suppressor genes A proto-oncogene is a normal cellular gene, that is associated with cancer only if it is improperly activated (usually by overexpression, mutation, or gene translocation). An oncogene is a mutant (activated) or overexpressed version of the proto-oncogene, found in cancer. A tumor suppressor gene only causes cancer if it is inactivated or deleted. In this way, it can be considered the opposite of an oncogene. An oncogene usually encourages cell growth, while a tumor suppressor gene usually inhibits cell growth For an oncogene, mutations that involve activation cause cancer For a tumor suppressor gene, mutations that cause inactivation cause cancer For oncogenes, mutations (activating) on one allele are sufficient to cause cancer, whereas for tumor suppressor genes, mutations (inhibiting) must occur on both alleles 5 Some of the ways oncogenes can be activated (activation of the proto-oncogene) • Point mutation that leads to constitutive activation of the gene product (the protein) • Mutations that cause the gene product to be more stable • Deletion of part of the gene, such a part the encodes a regulatory domain • Overexpression of the gene (too much transcription of the gene) • Gene amplification (excess copies of the DNA encoding the gene) 6 Viruses and early studies of oncogenes Although viruses cause only a minority of human cancers, viruses called tumor viruses led to the discovery of oncogenes, and were important for understanding cancer 7 Src and the importance of viruses in the identification of oncogenes 1909: Payton Rous removed a sarcoma from a chicken, ground it up, and filtered it through a fine filter. The filtrate was injected into other chickens, and resulted in tumors. Because of the fine filtration, the transmissible element was determined to be a virus. 8 The RSV retrovirus The virus was found to be a chicken RNA Virus (retrovirus) It is named Rous Sarcoma Virus (RSV) 9 RSV could lead to transformation in cultured cells Once the RSV virus was isolated, it was studied in cultured cells. Normal chicken fibroblasts were infected with the virus in a culture dish. A portion of these cells became transformed, which was evident by the development of foci. 10 The gene encoding Src was identified as the oncogenic portion of the RSV virus The part of this virus that led to the foci and to cancer was identified as a gene which was named v-Src. The researchers discovered that this was not a viral gene originally, but a gene that the virus picked up from a host cell during the virus’ lifecycle v-Src was found to be similar but not identical to a normal cellular gene: c-Src ==== v-Src = viral Src : An oncogene c-Src = cellular Src : A proto-oncogene 11 The lifecycle of a typical RNA Virus (Figure made with biorender) 12 How the RSV virus developed and incorporated v-Src ALV is now known to have been the original virus that infected the chicken cells During the infection, ALV acquired the Src gene, and this gave rise to the new virus: RSV The Src gene it incorporated was slightly different from the original cellular Src (c-Src) This difference is due to a small deletion in the gene sequence. Now revered to as v-Src RNA Original ALV virus New RLV virus (ALV: Avian Leukosis Virus, RSV: Rous Sarcoma Virus) 13 ALV and RSV After incorporating the Src gene, the ALV virus becomes the RSV virus v-Src (ALV: Avian Leukosis Virus, RSV: Rous Sarcoma Virus) 14 What is the normal function of Src? c-Src is a non-receptor tyrosine kinase. It has various roles in signaling pathways within the cell. It has roles in cell proliferation, differentiation, adhesion, migration,…. c-Src mediates its effects through phosphorylation of other signaling molecules in signal transduction pathways c-Src can be activated by stimuli such as growth factors, cytokines, and ECM proteins. Src can be activated by different mechanisms, including binding to cell surface receptors, including RTKs Activation of c-Src leads to a conformational change in Src that exposes its catalytic domain, thus allowing it to phosphorylate its targets Once activated it can activate pathways such as the MAP Kinase pathway, the AKT pathway, and the JAK/STAT pathway. Dysregulation of Src is now known to be associated with cancer 15 Functions of c-Src: In normal cells, c-Src can bind activated in different ways. One way it can get activated is by binding to RTKs at phospho-Tyrosine, through their SH2 domains ligand Src 16 Once activated, c-Src activates signaling pathways (Figure from Jiao, Q. et al. 2018) 17 c-Src compared to v-Src: c-Src is tightly regulated, while v-Src is constitutively active. C-Src is regulated by intramolecular interactions. In contrast, v-Src is truncated: It is and missing a stretch of DNA with an important tyrosine residue. Without this tyrosine residue Src can no longer form a closed, inactive conformation c-Src v-Src c-Src in an inactive conformation Krishnan et al. 2012 18 Summary: Regulation of c-Src (inactive) (inactive) B. PDGF receptor (an RTK) gets activated by binding to ligand. Thus becomes phosphorylated on tyrosine A. Inactive Src (Sh2 domain is bound to its own phospho-tyrosine) (missing in v-Src) Src catalytic domain (kinase domain) (Figures from Weinberg, the Biology of Cancer (active) C. The SH2 domain of the Src now binds to phospho-tyrosine on the PDGF receptor. (and the SH3 domain binds a proline rich region) This and several other steps relieves the inhibitory conformation and the catalytic domain is activated 19 Some history of the discovery of RSV and v-Src 1. Peyton Rous (1911): Discovered Rous Sarcoma Virus (RSV) that induces chicken tumors. 2. Howard Temin (1960s): Proposed the RNA tumor virus hypothesis, suggesting RNA viruses can cause cancer. 3. Temin (1970): Discovered reverse transcriptase, an enzyme retroviruses use to convert RNA into DNA. 4. Harold Varmus and Michael Bishop (1970s-1980s): Investigated src gene, a cellular proto-oncogene present in normal cells. 5. Varmus and Bishop: Found that viral oncogene v-src (from RSV) is derived from the cellular c-src gene. 6. Varmus and Bishop (1989): Awarded Nobel Prize in Physiology or Medicine for their work on oncogenes. 7. Rous, Temin, Varmus, and Bishop: Established the concept of oncogenes as genes involved in cancer development. 20 The importance of the discovery of v-Src Later, other oncogenes were found in other retroviruses Some oncogenes were also found in DNA viruses. Most oncogenes, however, are not viral in origin The work on Src was important because it led to the understanding that oncogenes could develop from normal cellular genes Abnormal activation or expression of Src is now known to occur in several human cancers. 21 Identification of new oncogenes Work on v-Src led to an interest in identifying new proto-oncogenes in human cells An assay was developed to identify human oncogenes: -DNA was isolated from human tumors -This DNA was broken into fragments -The DNA was then introduced into non-tumor mouse cells -Occasionally, the mouse cells became transformed: ie - they took on characteristics of cancer cells, and colonies of such cells grew. Each colony was a clone from a single cell DNA was isolated from the transformed colonies, and the human DNA was identified This was done by screening for human genome DNA specific repeat elements, such as Alu sequences 22 Ras This type of work contributed to the discovery of the Ras oncogene. Oncogenic Ras is one of the most well known oncogenes. Studies with Ras showed that a single mutation can cause normal cellular Ras to become oncogenic. The Ras gene (or one of the three Ras family members) is mutated in about 30% of human cancers. The three Ras family members are H-Ras, N-Ras, and K-Ras In addition to Src, it was one of the first oncogenes discovered 23 Normal Ras Inactive Ras GDP GEF (when bound to GTP, Ras is active. It takes on a conformational shape that allows it to interact with target proteins, such as Raf) GAP Ras GTP Active GAP: GTPase Activating Protein GEF: Guanine Nucleotide Exchange Factor Figure 5.30 The Biology of Cancer (Weinberg) 24 Normal Ras Ras is a molecular switch (it switches between bound GDP and bound GTP). It has some intrinsic GTPase activity, but this is inefficient on its own: Inactive Ras GDP GAP Ras GTP Active GEF (a GTPase is an enzyme that catalyzes the hydrolysis of GTP to GDP) GAP: A GAP is a GTPase Activating Protein. It can bind Ras and stimulate the GTPase activity of Ras, so that when GAP is present, GTP hydrolysis to GDP is efficient GEF: A GEF is a Guanine Nucleotide Exchange Factor. It exchanges GDP for GTP on Ras 25 Oncogenic Ras Oncogenic H-Ras has a single point mutation, compared with wild-type Ras (the proto-oncogene). This point mutation (originally found in bladder cancer) causes Ras to remain in the GTP bound state, by interfering with the intrinsic GTPase activity of Ras. Figure 4.10 The Biology of Cancer (Weinberg) 26 Oncogenic Ras remains GTP bound Point mutations have been found in all three major Ras family members. They cause Ras to be constitutively active (GTP bound). Most often this is via disruption of the intrinsic GTPase activity of Ras Figure 5.30 The Biology of Cancer (Weinberg) 27 Ras Once activated, Ras can activate multiple pathways in the cell, many of these are involved in cell growth / proliferation / cell cycle progression 28 Human cancers with Ras mutations Ras mutations are among the most common mutations in human cancers. The different Ras genes are K-Ras, N-Ras, H-Ras. K-Ras mutations are found at a rate of about 30%-50% in colorectal cancer, lung adenocarcinoma, pancreatic ductal adenocarcinoma. N-Ras mutations are often found in melanoma, acute myeloid leukemia (AML), and thyroid cancer. (10-25%) H-Ras mutations are less common. They are found in head and neck squamous cell carcinoma (HNSCC) and urinary bladder carcinoma. (510%). 29 Targeting Ras in human cancers: Targeting Ras has been challenging, possibly because of its important roles in many cellular functions. Lumakras (sotorasib): A small molecule inhibitor that targets mutant K-RAS (G12C) in non-small cell lung cancers (NSCLC). This mutation is found in about 13% of NSCLCs. Promising results were obtained in clinical trials, and the drug received accelerated FDA approval in 2021. 30 Other proto-oncogenes and oncogenes Myc 31 Cellular Myc Myc is a transcription factor that plays important roles in regulating cell growth, differentiation, and apoptosis Myc forms a heterodimer with another protein: Max The Myc/Max heterodimer binds to DNA sequences called E-boxes, and regulate transcription of various genes (Huang, H. et al. 2014) Myc is a transcription factor 32 The Myc oncogene Cellular Myc has a role in stimulating cell proliferation Improper activation of Myc can cause it to become an oncogene. Generally, improper activation of the Myc gene involves its overproduction. This can be due to: DNA amplification: DNA amplification, errors in DNA replication can lead to extra copies of the gene. Point mutations: Mutations in the gene can stabilize the encoded protein, which otherwise turns over rapidly. Other mutations may affect its interaction with Max Change in a regulatory element that controls Myc gene expression: For example, a chromosomal translocation can put a strong regulatory element next to the myc gene. Burkett’s lymphoma is an example of a cancer that involves such a translocation Myc is an important oncogene in several cancers, but directly targeting Myc with drugs remains challenging. 33 Myc in Burkitt’s lymphoma Because of a chromosomal translocation, the myc gene is placed under the transcriptional control of a strong enhancer for an immunoglobulin gene 34 Receptor Tyrosine Kinase (RTK) that can be oncogenes, including EGF Receptor and Her2/Neu 35 Mutations in Receptor Tryosine Kinases can cause them to be oncogenic (a) (b) (c) Oncogenic form of the receptor In this example (c) the EGF receptor is activated by truncation, resulting in an oncogenic form of the receptor: EGFRvIII This truncated form can become activated even without ligand binding This is a frequent mutation in glioblastoma (a brain cancer). (Figure from Alberts) It is a target for immunotherapy 36 Some targeted therapies for treating glioblastomas that have the EGFRvIII mutation (approved or under investigation): EGFR Inhibitors: Iressa (gefitinib), Tarceva (erlotinib), and Gilotrif (afatinib). These inhibitors target the wild-type EGFR, but can also inhibit EGFRvIII signaling. Vaccines: rindopepimut is a vaccine that is designed to stimulate the patient's immune system to attack cells expressing EGFRvIII. It is being tested, but is not yet an FDA approved treatment. Antibody-Drug Conjugates (ADCs): This type of targeted therapy combines an antibody with a cytotoxic drug. The antibody component recognizes EGFRvIII on tumor cells, delivering the cytotoxic drug to the cancer cells. Several EGFRvIII-targeted ADCs are currently under investigation. CAR-T Cell Therapy: In this therapy a patient's own T cells are modified so that they recognize EGFRvIII. Some early studies have been promising. 37 Her2/Neu is also an RTK, and has an important role in cancer - HER2 is part of the EGF receptor (EGFR) family. - This family consists of (EGFR)/HER1, HER2, HER3, and HER4 - The HER2 extracellular domain has no known ligand. - Instead, Her2 is normally activated by forming dimers with other members of the EGFR family. Schlam and Swain 2021 - Once it dimerizes it leads to activation of signaling pathways such as the MAP Kinase pathway and others, which can lead to cell proliferation. 38 Her2/Neu is also an RTK, and has an important role in cancer - HER2 is oncogenic when it is overexpressed or if its gene is amplified. This is associated with breast cancer. - Several drugs have been developed to block Her2 signaling in breast cancer - Some of these drugs also block other members of the EGFR family and may have relevance to other cancers. Schlam and Swain 2021 Her2 is an important oncogene in breast cancer 39 FGF receptor and bile duct cancer Cholangiocarcinomas (CCAs) are group of bile duct cancers. These include: -intrahepatic cholangiocarcinoma (iCCA), which arises in the liver, and -extrahepatic cholangiocarcinoma (eCCA), which arises from cells outside of the liver. iCCA has an especially poor prognosis. Fibroblast Growth Factor Receptor (FGFR), an RTK, has been implicated in iCCA. There are four FGFRs in human cells. The gene for one of these, FGFR2, is altered in some forms of iCCAs. The FGFR gene can be altered in various ways, including activating mutations, chromosomal translocations, gene fusions, and gene amplifications. This can result in ligand-independent constitutive activation of signaling pathways Lytgobi (futibatinib) is a new drug used to treat some patients with iCCA. It is a small molecule kinase inhibitor of FGFR. It inhibits FGFR2 by covalently binding to it. It was FDA approved in 9/30/22 40 FGF receptor and bile duct cancer Futibatinib and other FGFR inhibitors used to treat bile duct cancers Zugman, M. 2022 41 Raf Ligand (dimers) B-Raf Receptors (dimers) Raf and the MAP Kinase pathway (figure created with biorender) 42 B-Raf The three different Raf proteins in human cells are: A-Raf, B-Raf, and C-Raf (Raf-1) They are serine/threonine kinases with important roles in the MAP Kinase and other pathways. B-RAF is the most well studied and most commonly associated with cancer. B-RAF is frequently mutated in cancer, especially in melanoma The most common BRAF mutation in cancer is V600E (a substitution of valine for glutamic acid at position 600) This point mutation is found in approximately 50% of melanomas, and also some other cancers. This point mutation causes B-RAF to be constitutively active, regardless of the presence or absence of extracellular signals Drugs that inhibit Raf include Zelboraf (vemurafenib) and Tafinlar (deprafenib). They are used to treat some patients with melanoma 43 PI3K The normal function of PI3K: (Figure from Alberts) 44 PI3K PI3Kinase consists of two domains: A catalytic domain (PI3KCA, also referred to as p110) and a regulatory domain (PI3KR1, also referred to as p85) (Geering, B. et al. 2007) Inhibited state Activated state p85, the regulatory subunit, binds to and inhibits p110, the catalytic subunit. However, when p85 binds to phospho-tyrosines (as on RTKs), this relieves its inhibitory effect on p110. P110 can now phosphorylate its substrate (PIP2) 45 PI3KCA Mutations in PIK3CA (p110) have been found in several human cancers including breast, colorectal, ovarian, brain, and other cancers. Mutations are often point mutations. PIK3CA mutations have been found in about 25% of breast cancers, and are associated with a poorer prognosis compared with some other breast cancers These mutations lead to dysregulation of pathways such as the PI3K/AKT/mTOR pathway, leading to uncontrolled cell growth and survival. Some drugs that target the PI3K/AKT/mTOR pathway may be promising for treating these cancers. 46 PI3KCA (p110) mutations The most common PI3K mutations are in two ‘hotspots’ in the PI3KCA (p110) gene, specifically in exons 9 and 20. The mutations in these hotspots account for about 80-90% of all PIK3CA mutations in human cancers These mutations include: H1047R (substitution of histidine for arginine) and E545K, E545K (substitution of glutamic acid for lysine) Catalytic domain Echelon-inc.com Regulatory domain The E545K mutant may disrupt the inhibition of p110 by p85, The exact mechanism for the other mutations may have different functions and are not thoroughly understood. 47 Some PI3K inhibitors for cancer treatment Zydelig (idelalisib) targets the catalytic subunit of an isoform of PI3K. It is approved for some types of blood cancer, in patients who have previously received other treatments. FDA approved in 2014 Aliqopa (copanlisib) is FDA approved for treatment of follicular lymphoma (a white blood cell cancer) in 2017 Several other PI3K inhibitors have been FDA approved more recently, some of which are used in combination with other treatments. 48 49 CDK4 Rb DP1 P M Rb E2F DP1 Rb G2 growth factors E2F G1 Go CyclinD Rb P CyclinD Cdk4/6 S Cdk4/6 Rb restriction point DP1 E2F P Rb P Rb interphase + E2F transcription repressed DP1 DP1 E2F transcription 50 CDK4 CDK4 has an important role in regulating the cell cycle and allowing progression to S phase CDK4 gene amplification is commonly found in some types of cancers, especially melanoma and some sarcomas, and in some cases in breast, lung, and bladder cancers. While the most common mechanism of CDK4 in cancer is gene amplification, CDK4 can sometimes also be regulated by activating mutations. These mutations are in the kinase domain, leading to its constitutive activation. These activating mutations are most commonly found in melanoma 51 CDK4/6 inhibitors in cancer Early studies of CDK4/6 inhibitors were unsuccessful. Several newer inhibitors, however, are more promising. Ibrance (palbociclib) (Pfizer), Kisqali (ribociclib) (Novartis), and Verzenio (abemaciclib) (Lilly) are CDK4/6 inhibitors administered orally, and approved as therapy for advanced HR-positive Her2 negative breast cancer Cosela (Trilaciclib), is a CDK4/6 inhibitor given by IV injection, It has been approved for small cell lung cancer and other cancers. It is also used as a myelopreservation therapy. It does so by temporarily halting the growth of hematopoietic stem cells during chemotherapy, thus protecting them from toxic effects of the therapy. Other inhibitors are also at various stages of development (myelo: bone marrow) Goel, S., et al. 2022) 52 Resources Helpful Reading Material Alberts. Molecular Biology of the Cell, Chapter 20 Goel, S. et al. (2022). Targeting CDK4 and CDK6 in cancer. Nat Rev Cancer 22(6):356-372 References for some of the figures Weinberg, R. The Biology of Cancer, Chapter 3 Liu, W. et al. (2015). The molecular effect of metastasis suppressors on Src signaling and tumorigenesis: new therapeutic targets. Oncotarget 6: 35522-35541 Jiao, Q. et al. (2018). Advances in studies of tyrosine kinase inhibitors and their acquired resistance. Molecular Cancer. 17:36 Bunz, F. (2016). Principles of Cancer Genetics. Springer Huang, H. (2014). Attacking c-Myc: Targeted and Combined Therapies for Cancer. Current Pharmaceutical Design, 2014, 20, 6543-6554 Schlam, I, and Swain, S. (2021). HER2-positive breast cancer and tyrosine kinase inhibitors: the time is now. NPJ Breast Cancer. 2021 May 20;7(1):56 Zugman, M. (2022). Precision Medicine Targeting FGFR2. Genomic Alterations in Advanced Cholangiocarcinoma: Current State and Future Perspectives. Frontiers in Oncology. 12: 860453 Geering, B. (2007). Regulation of class IA PI3Ks: is there a role for monomeric PI3K subunits? chem Soc Trans. 35(Pt 2):199-203 53