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Dec 9, 2011

Scientists Devise Sideways Approach to Killing Myc-Addicted Tumors

  • Inhibiting SUMOylation in Myc oncogene-carrying tumor cells could represent a new approach to treating cancers that are driven by Myc hyperactivation, researchers claim. A Baylor college of Medicine and Harvard Medical School-led team used a genome-wide RNAi screen to search for genes that were vital to supporting Myc-addicted tumors. Their screening results and further in vitro and in vivo tests found that inactivating SUMO-activating enzyme (SAE1/2) function led to catastrophic spindle defects upon Myc hyperactivation, which resulted in apoptosis.

    Essentially, the SUMO-activating enzyme is vital to enable cells to tolerate Myc hyperactivation, Baylor’s Thomas F. Westbrook, M.D., and colleagues claim. "Loss of SUMOylation leads to substantial mitotic catastrophe and cell death by switching a subprogram of Myc transcriptional targets that support mitotic spindle function."

    Gene-expression studies in human breast cancer patients in addition found that low SAE1/SAE2 levels in Myc-high tumors correlated with longer metastatic-free survival in patients. The team reports its findings in Scienceexpress, in a paper titled “A SUMOylation-Dependent Transcriptional Subprogram Is Required for Myc-Driven Tumorigenesis.”

    The Myc gene codes for a transcription factor that is often dysregulated in cancer cells through amplification, mutation, overexpression, or protein stabilization, and in cancer cells that express the Myc oncogene are dependent on it for survival, a concept known as gene addiction.

    Attempts to develop anticancer therapies that block the Myc oncogene, however, have been hampered by the fact that the protein target lacks efficient efficient bindings sites for drug compounds. One potential alternative approach would be to identify a pathway that the Myc oncogene itself relies on, and that is itself therapeutically viable. This is a concept known as non-oncogene addiction (NOA). Unfortunately, genes and signaling pathways underlying oncogenic support processes are difficult to identify, as they are not in themselves oncogenes or mutated in cancer. As a result, NOA pathways supporting the Myc oncogene are poorly understood, the team notes.

    The researchers' approach to finding such pathways involved a genome-wide genetic screen for Myc-synthetic lethal (MySL) shRNAs in human mammary epithelial cells (HMECs) engineered with an inducible c-Myc-estrogen receptor fusion transgene (Myc-ER HMECs). In this system, induction of the transgene leads to increased expression of known Myc targets.

    The team used the system to screen nearly 75,000 shRNAs in order to identify those that led to cell death only in the presence of aberrant Myc signaling. Some 403 MySL shRNAs candidates were identified, and gene ontology analysis indicated that these were largely enriched for ion channels and ubiquitin-like protein conjugation, as well as components of the mitotic spindle. A number of the candidates had previously been implicated in the survival of Myc hyperactivated cells, as well as novel candidates. Interestingly, further analysis of the MySL candidates highlighted a highly connected protein-protein interaction network, in which many components were involved in the mitotic spindle, “suggesting that Myc hyperactivation may impose a stress on proper mitotic progression,” the team notes.

    The most significant of the candidates with previously unrecognized roles in Myc biology was the SUMO-activating enzyme subunit 2 (SAE2/UBA2), which is a critical component of the SUMO-activating enzyme needed for SUMO-conjugation to proteins. In fact multiple SAE2-shRNAs in the primary screen exhibited Myc synthetic lethality, as did shRNAs to SAE1, the heterodimeric partner of SAE2.

    Further studies using the Myc-ER HMECs confirmed that shRNAs targeting SAE2 did lead to depletion of the protein, and also resulted in markedly increased cell doubling time on Myc induction. The same results were obtained when a constitutive Myc transgene was expressed together with shSAE2, while two independent SAE2 shRNAS produced a Myc-synthetic lethal phenotype. “Multiple shRNAs targeting SAE1 and the downstream SUMO E2 conjugating enzyme UBE2I (UBC9) were also synthetically lethal with Myc hyperactivation, demonstrating that SUMOylation interference is synthetically lethal with hyperactivated Myc,” the team notes.

    The depletion of SAE2 was associated with decreased levels of SUMO1- or SUMO2/3-modified proteins, suggesting the deficit resulted in a global SUMOylation impairment in these cells. Conversely, restoring wild-type SAE2 suppressed the MySL phenotype of SAE2 shRNA.

    The next obvious question was, how does SAE2 depletion in the presence of Myc hyperactivation impair cell proliferation? They found that in SAE2-depleted cells, Myc-induction led to increases in G2/M DNA content and aberrant  (>2N) content, characteristic of mitotic defects that are known to lead to apoptosis, which ties in with the earlier screen results of an enrichment for genes involved in the mitotic spindle. This was supported by the observation that Myc-active/SAE2-inactive HMECs exhibited significantly more spindle defects than cells expressing Myc-alone or shSAE2-alone.

    Gene-expression profiling indicated that SAE2 depletion led to repression of 22.5% of the 605 Myc-induced mRNA transcripts, and the team termed these genes SUMOylation-dependent Myc-switchers (SMS genes). Again, further analyses indicated that SMS genes were significantly enriched for regulators of the mitotic spindle, and 17 of the 86 SMS genes have previously been shown to participate in the assembly or integrity of mitotic spindles. “These observations highlight regulation of spindle assembly as a key vulnerability in cells harboring the Myc-active/SAE2-inactive state, and suggest that SMS genes may be linchpins in the Myc-SAE2 synthetic lethal relationship,” the authors write. In support of this notion, the team found that three of the top four SMS genes (CASC5, BARD1, and CDC20) were synthetically lethal with Myc hyperactivation. Importantly, depleting SAE2 in human Myc-dependent breast cancer cells led to a reduction in cell growth.

    To test this reliance of human breast cancer cells on SAE2 in vivo, the team engineered Myc-dependent and Myc-independent breast cancer cells engineered with a dox-inducible SAE2-shRNA, and transplanted the cells into immunocompromised mice. Depletion of SAE2 inhibited growth of the Myc-dependent but not the Myc-independent tumors, and also increased the survival times of animals carrying the SAE2-depleted Myc-dependent growths.

    The data generated led the researchers to hypothesize whether Myc-high human breast cancers that expressed low levels of SUMO-activating enzyme might be less aggressive. When they compiled breast cancer datasets to investigate this possibility, they found that patients with Myc-high tumors who also demonstrated low-level expression of SAE1/SAE2 had significantly better metastasis-free survival than those with higher SAE1/SAE2. However, lower-level expression of SAE1/SAE2 didn’t correlate with survival in patients with Myc-low tumors.

    “We have shown that E1 SUMO-activating enzyme enables cells to tolerate Myc hyperactivation,” the investigators conclude. “inactivation of SAE2 mimics the mitotic disruption caused by spindle poisons, but in a genotype-specific way (i.e., selectively in cells that harbor oncogenic Myc activation).” Mitotic interference is already a key approach to cancer therapy, but while relevant drugs such as taxanes can be used to treat a range of cancers, these types of therapeutics are also toxic to healthy tissues. “Our observation that inhibition of SUMOylation can mimic spindle poisons selectively in cells expressing hyperactivated Myc raises the possibility that drugs targeting the SUMO pathway may have the antitumor effects of spindle poisons with fewer side effects.”


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