Loss of function RNA interference screens for molecular targets in cancer

SY27-02 We have designed a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We are using this “Achilles heel” screen to identify those cellular proteins and pathways that are most vulnerable to therapeutic attack. A doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) was used to construct a library targeting 2,500 human genes, with each gene targeting by 3-6 shRNAs. Each vector was engineered to contain a unique 60-base pair “bar code”, allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar code sequences. In a typical screen, retroviral pools of 500-1000 vectors from this library are used to a infect cancer cell line. After selection for stable integration, doxycycline is added to half of the culture to induce shRNA expression, and the remaining half serves as an uninduced control. These two cultures are then grown in parallel for 3 weeks, during which time cells expressing toxic shRNAs will be depleted from the induced culture. DNA bar code microarrays are used to compare the abundance of each shRNA vector in the induced versus uninduced populations, thereby revealing which shRNAs inhibited the proliferation or survival of the cancer cells. In our genetic screens, we have use a “synthetic lethal” strategy in which we search for shRNAs that are only toxic to cancer types with particular underlying genetic lesions. To this end, we conducted parallel screens in two distinct molecular subgroups of diffuse large B cell lymphoma (DLBCL) termed activated B cell-like (ABC) DLBCL and germinal center B cell-like (GCB) DLBCL. These two subgroups are profoundly different in their pathogenesis in that they arise from different stages of normal B lymphocyte development and utilize distinct oncogenic mechanims. Notably, we had previously discovered that the ABC DLBCL subgroup depends upon the NF-kB pathway for survival, but the GCB DLBCL subgroup does not. However, we did not know which upstream signaling pathways in ABC DLBCLs were responsible for their constitutive activaty of the IkB kinase, the key regulator of the NF-kB pathway. Remarkably, shRNAs targeting the NF-kB pathway were depleted in screens of ABC DLBCL but not GCB DLBCL cells, in keeping with the essential role of this pathway in the survival of ABC DLBCL. Unexpectedly, this screen uncovered an upstream signaling pathway involving CARD11, MALT1 and BCL10 that is responsible for the constitutive NF-kB activity in ABC DLBCL. Knock down of any of these genes with shRNAs descreased IkB kinase activity, NF-kB target gene expression, and cell viability. The CARD11 pathway therefore represents an attractive new therapeutic target for the ABC DLBCL subgroup. Importantly, therapeutic targeting of this pathway is likely to have discrete and manageable toxicity since mice that are deficient in these genes are viable and only have defects in subsets of B and T lymphocytes. More recently, we have used the Achilles heel methodology to discover genes essential for cellular proliferation and survival in other forms of lymphoid malignancy. We believe that a new taxonomy of cancer may emerge in which cancers are defined by their critical dependence on particular signaling pathways for survival and proliferation. Another important use of this technology will be to evaluate which of the many somatically mutated genes in human cancer make the most important ongoing contributions to the biology of cancer cells. Finally, these genetic screens are revealing that cancers may “inherit” from their normal cellular counterparts a dependance on particular regulatory pathways for their proliferation and survival. These pathways may not necessary be affected by genetic alterations but the cancer cells may nevertheless be “addicted” to their continous activity. Therefore, this methodology has the potential to uncover a new class of therapeutic targets distinct from known oncogenes.