GigaAssay - An adaptable high-throughput saturation mutagenesis assay platform.

High-throughput assay systems have had a large impact on understanding the mechanisms of basic cell functions. However, high-throughput assays that directly assess molecular functions are limited. Herein, we describe the "GigaAssay", a modular high-throughput one-pot assay system for measuring molecular functions of thousands of genetic variants at once. In this system, each cell was infected with one virus from a library encoding thousands of Tat mutant proteins, with each viral particle encoding a random unique molecular identifier (UMI). We demonstrate proof of concept by measuring transcription of a GFP reporter in an engineered reporter cell line driven by binding of the HIV Tat transcription factor to the HIV long terminal repeat. Infected cells were flow-sorted into 3 bins based on their GFP fluorescence readout. The transcriptional activity of each Tat mutant was calculated from the ratio of signals from each bin. The use of UMIs in the GigaAssay produced a high average accuracy (95%) and positive predictive value (98%) determined by comparison to literature benchmark data, known C-terminal truncations, and blinded independent mutant tests. Including the substitution tolerance with structure/function analysis shows restricted substitution types spatially concentrated in the Cys-rich region. Tat has abundant intragenic epistasis (10%) when single and double mutants are compared.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53.

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

MCL-1 but not BCL-XL is critical for the development and sustained expansion of thymic lymphoma in p53-deficient mice.

Apoptosis plays a role in normal lymphopoiesis and lymphoid malignancies. Pro-survival MCL-1 is essential for survival of T-cell progenitors, BCL-XL for immature thymocytes, and BCL-2 for mature T cells. Conversely, little is known about the regulators that are required for the survival of T-cell lymphomas. We used constitutive and conditionally gene-targeted mice to investigate which pro-survival BCL-2 family member is required for the sustained survival of thymic lymphomas initiated by loss of p53. Constitutive loss of a single Mcl-1 allele delayed tumor onset. In contrast, lymphomas emerging in p53(-/-) mice in which Mcl-1 could be conditionally deleted had been selected for retention of MCL-1 expression. In contrast, complete loss of BCL-XL had no impact on lymphoma development in p53(-/-) mice. These results demonstrate that thymic lymphomas elicited by loss of p53 must arise from cancer-initiating cells that require MCL-1 for their survival. Acute deletion of both Mcl-1 alleles abrogated the expansion of p53(-/-) lymphomas in mice, whereas inducible loss of BCL-XL had little impact. This reveals that MCL-1 is essential for the sustained survival of these malignant cells and suggests that targeting MCL-1 may be an attractive strategy for the treatment of T-cell lymphoma.

Data supporting a saturation mutagenesis assay for Tat-driven transcription with the GigaAssay.

The data in this article are associated with the research paper "GigaAssay - an adaptable high-throughput saturation mutagenesis assay" [1]. The raw data are sequence reads of HIV-1 Tat cDNA amplified from cellular genomic DNA in a new single-pot saturation mutagenesis assay designated the "GigaAssay". A bioinformatic pipeline and parameters used to analyze the data. Raw, processed, analyzed, and filtered data are reported. The data is processed to calculate the Tat-driven transcription activity for cells with each possible single amino acid substitution in Tat. This data can be reused to interpret Tat intermolecular interactions and HIV latency. This is one of the largest and most complete datasets regarding the impact of amino acid substitutions within a single protein on a molecular function.

P53 orchestrates a complex symphony of cellular processes during oncosuppression.

We recently showed that the p53 tumor suppressor simultaneously governs numerous cellular processes in one model of transformation suppression. These findings suggest that p53-mediated tumor suppression relies on coordinated modulation of diverse cellular functions in a particular context, helping to explain why loss of the TP53 (tumor protein p53) gene is so prevalent in human cancers.

Polymerase delta-interacting protein 38 (PDIP38) modulates the stability and activity of the mitochondrial AAA+ protease CLPXP.

Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/ß linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like ß-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like ß-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.

p53 deficiency triggers dysregulation of diverse cellular processes in physiological oxygen.

The mechanisms by which TP53, the most frequently mutated gene in human cancer, suppresses tumorigenesis remain unclear. p53 modulates various cellular processes, such as apoptosis and proliferation, which has led to distinct cellular mechanisms being proposed for p53-mediated tumor suppression in different contexts. Here, we asked whether during tumor suppression p53 might instead regulate a wide range of cellular processes. Analysis of mouse and human oncogene-expressing wild-type and p53-deficient cells in physiological oxygen conditions revealed that p53 loss concurrently impacts numerous distinct cellular processes, including apoptosis, genome stabilization, DNA repair, metabolism, migration, and invasion. Notably, some phenotypes were uncovered only in physiological oxygen. Transcriptomic analysis in this setting highlighted underappreciated functions modulated by p53, including actin dynamics. Collectively, these results suggest that p53 simultaneously governs diverse cellular processes during transformation suppression, an aspect of p53 function that would provide a clear rationale for its frequent inactivation in human cancer.

DNA repair processes are critical mediators of p53-dependent tumor suppression.

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development.

A p53 Super-tumor Suppressor Reveals a Tumor Suppressive p53-Ptpn14-Yap Axis in Pancreatic Cancer.

The p53 transcription factor is a critical barrier to pancreatic cancer progression. To unravel mechanisms of p53-mediated tumor suppression, which have remained elusive, we analyzed pancreatic cancer development in mice expressing p53 transcriptional activation domain (TAD) mutants. Surprisingly, the p5353,54 TAD2 mutant behaves as a "super-tumor suppressor," with an enhanced capacity to both suppress pancreatic cancer and transactivate select p53 target genes, including Ptpn14. Ptpn14 encodes a negative regulator of the Yap oncoprotein and is necessary and sufficient for pancreatic cancer suppression, like p53. We show that p53 deficiency promotes Yap signaling and that PTPN14 and TP53 mutations are mutually exclusive in human cancers. These studies uncover a p53-Ptpn14-Yap pathway that is integral to p53-mediated tumor suppression.

Therapeutic Response to Non-genotoxic Activation of p53 by Nutlin3a Is Driven by PUMA-Mediated Apoptosis in Lymphoma Cells.

Nutlin3a is a small-molecule antagonist of MDM2 that promotes non-genotoxic activation of p53 through p53 protein stabilization and transactivation of p53 target genes. Nutlin3a is the forerunner of a class of cancer therapeutics that have reached clinical trials. Using transgenic and gene-targeted mouse models lacking the critical p53 target genes, p21, Puma, and Noxa, we found that only loss of PUMA conferred profound protection against Nutlin3a-induced killing in both non-transformed lymphoid cells and Eµ-Myc lymphomas in vitro and in vivo. CRISPR/Cas9-mediated targeting of the PUMA gene rendered human hematopoietic cancer cell lines markedly resistant to Nutlin3a-induced cell death. These results demonstrate that PUMA-mediated apoptosis, but not p21-mediated cell-cycle arrest or senescence, is a critical determinant of the therapeutic response to non-genotoxic p53 activation by Nutlin3a. Importantly, in human cancer, PUMA expression may predict patient responses to treatment with MDM2 antagonists.

p53 efficiently suppresses tumor development in the complete absence of its cell-cycle inhibitory and proapoptotic effectors p21, Puma, and Noxa.

Activation of apoptosis through transcriptional induction of Puma and Noxa has long been considered to constitute the critical (if not sole) process by which p53 suppresses tumor development, although G1/S boundary cell-cycle arrest via induction of the CDK inhibitor p21 has also been thought to contribute. Recent analyses of mice bearing mutations that impair p53-mediated induction of select target genes have indicated that activation of apoptosis and G1/S cell-cycle arrest may, in fact, be dispensable for p53-mediated tumor suppression. However, the expression of Puma, Noxa, and p21 was not abrogated in these mutants, only reduced; therefore, the possibility that the reduced levels of these critical effectors of p53-mediated apoptosis and G1/S-cell-cycle arrest sufficed to prevent tumorigenesis could not be excluded. To resolve this important issue, we have generated mice deficient for p21, Puma, and Noxa (p21-/-puma-/-noxa-/- mice). Cells from these mice were deficient in their ability to undergo p53-mediated apoptosis, G1/S cell-cycle arrest, and senescence. Nonetheless, these animals remained tumor free until at least 500 days, in contrast to p53-deficient mice, which had all succumbed to lymphoma or sarcoma by 250 days. Interestingly, DNA lesions induced by γ-irradiation persisted longer in p53-deficient cells compared to wild-type or p21-/-puma-/-noxa-/- cells, and the former failed to transcriptionally activate several p53 target genes implicated in DNA repair. These results demonstrate beyond a doubt that the induction of apoptosis, cell-cycle arrest, and possibly senescence is dispensable for p53-mediated suppression of spontaneous tumor development and indicate that coordination of genomic stability and possibly other processes, such as metabolic adaptation, may instead be critical.

The role of the apoptotic machinery in tumor suppression.

Multicellular organisms have evolved processes to prevent abnormal proliferation or inappropriate tissue infiltration of cells, and these tumor suppressive mechanisms serve to prevent tissue hyperplasia, tumor development, and metastatic spread of tumors. These include potentially reversible processes such as cell cycle arrest and cellular senescence, as well as apoptotic cell death, which in contrast eliminates dangerous cells that may initiate tumor development. Tumor suppressive processes are organized as complex, extensive signaling networks, controlled by central "nodes." These "nodes" are prominent tumor suppressors, such as P53 or PTEN, whose loss is responsible for the development of the majority of human cancers. In this review we discuss the processes by which some of these prominent tumor suppressors trigger apoptotic cell death and how this process protects us from cancer development.