Differentiation therapy and the mechanisms that terminate cancer cell proliferation without harming normal cells

Abstract

Chemotherapeutic drugs have a common intent to activate apoptosis in tumor cells. However, master regulators of apoptosis (e.g., p53, p16/CDKN2A) are frequently genetically inactivated in cancers, resulting in multidrug resistance. An alternative, p53-independent method for terminating malignant proliferation is to engage terminal-differentiation. Normally, the exponential proliferation of lineage-committed progenitors, coordinated by the master transcription factor (TF) MYC, is self-limited by forward-differentiation to terminal lineage-fates. In cancers, however, this exponential proliferation is disengaged from terminal-differentiation. The mechanisms underlying this decoupling are mostly unknown. We performed a systematic review of published literature (January 2007-June 2018) to identify gene pathways linked to differentiation-failure in three treatment-recalcitrant cancers: hepatocellular carcinoma (HCC), ovarian cancer (OVC), and pancreatic ductal adenocarcinoma (PDAC). We analyzed key gene alterations in various apoptosis, proliferation and differentiation pathways to determine whether it is possible to predict treatment outcomes and suggest novel therapies. Poorly differentiated tumors were linked to poorer survival across histologies. Our analyses suggested loss-of-function events to master TF drivers of lineage-fates and their cofactors as being linked to differentiation-failure: genomic data in TCGA and ICGC databases demonstrated frequent haploinsufficiency of lineage master TFs (e.g., GATA4/6) in poorly differentiated tumors; the coactivators that these TFs use to activate genes (e.g. ARID1A, PBRM1) were also frequently inactivated by genetic mutation and/or deletion. By contrast, corepressor components (e.g., DNMT1, EED, UHRF1, and BAZ1A/B), that oppose coactivators to repress or turn off genes, were frequently amplified instead, and the level of amplification was highest in poorly differentiated lesions. This selection by neoplastic evolution towards unbalanced activity of transcriptional corepressors suggests these enzymes as candidate targets for inhibition aiming to re-engage forward-differentiation. This notion is supported by both pre-clinical and clinical trial literature.

Fig. 1. MYC alterations across multiple human malignancies.

a TCGA and IGCG data were analyzed through cBioPortal to determine aberrations at the MYC locus using pre-assigned GISTIC scores in multiple cancers from different tissue types. b We analyzed TCGA PANCAN data sets available through TCGA hub in Xena Browser. Survival analysis of cases with copy number (CN) gains and amplification at the MYC loci vs. those with CN WT/minor deletion of MYC demonstrated a significant overall survival (p-value < 9.784E−11, LogRank test, n = 1352 WT/minor del, 2628 CN gain and amplification). Survival data analyzed in Xena Browser (https://xenabrowser.net/) c Anlysis of MYC GISTIC Score vs. MYC mRNA expression using PANCAN RNA-seq data available in TCGA hub in Xena Browser. There was a strong correlation with spearman r = 0.3339, p < 0.0001, n = 9697. d Survival analysis of patients with increased MYC mRNA compared to those with decreased MYC mRNA expression. Expression levels are normalized relative to expression levels in normal tissues. Increased MYC mRNA was associated with poor survival (n = 1762) compared to decreased MYC mRNA (n = 1776, p = 5.06 × 10⁻¹⁸ e Schematic representation of metazoan differentiation and how differentiation is stalled in malignant cells. Differentiation continuum is initiated through stem cells lineage commitment, followed by exponential proliferation of tissue precursors/progenitors mediated by two copies of the MYC gene. To maintain homeostasis, MYC-mediated proliferation is dominantly antagonized by terminal differentiation pathways. f Human malignancies have impaired differentiation that fails to antagonize the MYC gene allowing for exponential proliferation of tissue precursors

Fig. 2. Apoptosis induction in p53/p16 mutant malignancy remains toxic to normal cells while simultaneous linked to refractory disease.

a Data was downloaded from TCGA and ICGC and analyzed in cBioPortal for mutations in TP53 and CDKN2A genes. b Top 10 malignancies with high TP53/CDKN2A alterations (TP53/CDKN2A high). *Cases where these alterations were linked to poor disease-free or overall survival with a p-value < 0.05 (Table S1). c Bottom 10 cases with least frequency of alterations in TP53/CDKN2A (TP53/CDKN2A low). *Cases where these alterations were linked to poor disease-free or overall survival with a p-value ≤ 0.05 d Disease-free survival of testicular cancer, cases with minor alterations (gains, and heterozygous loss of one allele in TP53 and CDKN2A) vs. cases with wild type TP53 and CDKN2A (p-value = 0.211, LogRank test). e Disease-free survival of pancreatic cancer with mutant TP53 and CDKN2A cases was significantly lower vs. cases with wild-type TP53 and CDKN2A (p-value = 0.0078, LogRank test). f Disease-free survival of liver cancer was also significantly lower in mutant TP53 and CDKN2A cases vs. wild-type (p-value = 0.0068, LogRank test). g Quantitative analysis of TP53 and CDKN2A mutations demonstrated less frequency of alteration of these genes in curable vs. high refractory/treatment resistant human malignancies. h During physiologic maturation, unhealthy cells with WT p53/p16 undergo irreversible apoptosis. Alterations in these proteins sustain oncogenic evolution leading to aberrant proliferation without apoptosis

Fig. 3. Genetic alterations in lineage specifying master transcription factors in human malignancies.

a Analysis of TCGA data deposited in cBioPortal to determine alterations of master transcription factors of various lineages (Table 1). Key lineage specifying transcriptions factors were mostly haploinsufficient (heterozygous deletion/hetloss) in malignant cells or contained frequent amplification and gains. None of the transcription factors had biallelic frameshift inactivating mutations. Thus stalled differentiation occurs through genetic haploinsuffiency of key lineage specific transcription factors. b Analysis of FOXL1 deletions across varying degrees of differentiation grades (pathological grades) of ovarian cancer. c Analysis of GATA4 deletions across varying degrees of differentiation grades of pancreatic cancer (PDAC). d Analysis of GATA deletions across varying degrees of differentiation grades liver cancer (HCC)

Fig. 4. Frequent inactivating mutations of coactivators and amplification and copy number gains at gene loci of transcriptional corepressors.

TCGA data was analyzed in cBioPortal to determine frequent genetic alterations in transcriptional corepressor and coactivator enzymes (Table 1). a Inactivating mutations, bi-allelic and frameshift mutations and deletions of transcriptional coactivator enzymes in ovarian, pancreatic and liver cancers (Table 1). b Copy number (CN) gain and amplifications of corepressors was frequently observed in various tumors including ovarian cancer (OVC), pancreatic cancer (PDAC) and liver cancer (HCC) (Table 1). c Analysis of HES1 CN gains across varying degrees of differentiation grades (pathological grades) of OVC. d Analysis of CN gains of BAZ1B across varying degrees of differentiation grades PDAC. e Analysis of CN gains KDM1B gains across varying degrees of differentiation grades HCC

Fig. 5. Corepressor upregulation and model for inhibiting corepressors to re-engage forward-differentiation.

a Corepressor DNMT1 mRNA upregulation predicts poor survival across multiple human malignancies in TCGA PANCAN data. b Corepressor UHRF1 (that partners with DNMT1 for epigenetic repression activities) mRNA upregulation predicts poor survival across multiple human malignancies in TCGA PANCAN data. c Model example in PDAC alterations of coactivators and corepressors and candidate small molecules that can be used as corepressor therapy. d Model schematic summary for p-53 independent differentiation-restoring therapy. Non-malignant cells (normal cells) have intact lineage specifying transcription factors of cell fate determination that dynamically recruit coactivators and corepressors enzymes to turn on or turn off differentiation genes. Gene dose reduction by heterozygous deletion of a master transcription factor and inactivating mutations in its coactivators impairs the activation component of differentiation genes epigenetically. Aberrant amplifications in transcriptional corepressor enzymes facilitate a closed chromatin status and epigenetically silence hundreds of differentiation genes^, (Table 1). This mode of alteration is clinically relevant and can be developed to suppress proliferation even in TP53 mutant malignancies^,