Small-molecule targeted therapies induce dependence on DNA double-strand break repair in residual tumor cells.

Residual cancer cells that survive drug treatments with targeted therapies act as a reservoir from which eventual resistant disease emerges. Although there is great interest in therapeutically targeting residual cells, efforts are hampered by our limited knowledge of the vulnerabilities existing in this cell state. Here, we report that diverse oncogene-targeted therapies, including inhibitors of epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), KRAS, and BRAF, induce DNA double-strand breaks and, consequently, ataxia-telangiectasia mutated (ATM)-dependent DNA repair in oncogene-matched residual tumor cells. This DNA damage response, observed in cell lines, mouse xenograft models, and human patients, is driven by a pathway involving the activation of caspases 3 and 7 and the downstream caspase-activated deoxyribonuclease (CAD). CAD is, in turn, activated through caspase-mediated degradation of its endogenous inhibitor, ICAD. In models of EGFR mutant non-small cell lung cancer (NSCLC), tumor cells that survive treatment with small-molecule EGFR-targeted therapies are thus synthetically dependent on ATM, and combined treatment with an ATM kinase inhibitor eradicates these cells in vivo. This led to more penetrant and durable responses in EGFR mutant NSCLC mouse xenograft models, including those derived from both established cell lines and patient tumors. Last, we found that rare patients with EGFR mutant NSCLC harboring co-occurring, loss-of-function mutations in ATM exhibit extended progression-free survival on first generation EGFR inhibitor therapy relative to patients with EGFR mutant NSCLC lacking deleterious ATM mutations. Together, these findings establish a rationale for the mechanism-based integration of ATM inhibitors alongside existing targeted therapies.

Dysregulation of mitochondrial dynamics proteins are a targetable feature of human tumors.

Altered mitochondrial dynamics can broadly impact tumor cell physiology. Using genetic and pharmacological profiling of cancer cell lines and human tumors, we here establish that perturbations to the mitochondrial dynamics network also result in specific therapeutic vulnerabilities. In particular, through distinct mechanisms, tumors with increased mitochondrial fragmentation or connectivity are hypersensitive to SMAC mimetics, a class of compounds that induce apoptosis through inhibition of IAPs and for which robust sensitivity biomarkers remain to be identified. Further, because driver oncogenes exert dominant control over mitochondrial dynamics, oncogene-targeted therapies can be used to sensitize tumors to SMAC mimetics via their effects on fission/fusion dynamics. Collectively, these data demonstrate that perturbations to the mitochondrial dynamics network induce targetable vulnerabilities across diverse human tumors and, more broadly, suggest that the altered structures, activities, and trafficking of cellular organelles may facilitate additional cancer therapeutic opportunities.

A Landscape of Therapeutic Cooperativity in KRAS Mutant Cancers Reveals Principles for Controlling Tumor Evolution.

Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor-based combination therapy for KRAS/PIK3CA double-mutant colorectal cancers (CRCs) with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence.

Codon bias imposes a targetable limitation on KRAS-driven therapeutic resistance.

KRAS mutations drive resistance to targeted therapies, including EGFR inhibitors in colorectal cancer (CRC). Through genetic screens, we unexpectedly find that mutant HRAS, which is rarely found in CRC, is a stronger driver of resistance than mutant KRAS. This difference is ascribed to common codon bias in HRAS, which leads to much higher protein expression, and implies that the inherent poor expression of KRAS due to rare codons must be surmounted during drug resistance. In agreement, we demonstrate that primary resistance to cetuximab is dependent upon both KRAS mutational status and protein expression level, and acquired resistance is often associated with KRASQ61 mutations that function even when protein expression is low. Finally, cancer cells upregulate translation to facilitate KRASG12-driven acquired resistance, resulting in hypersensitivity to translational inhibitors. These findings demonstrate that codon bias plays a critical role in KRAS-driven resistance and provide a rationale for targeting translation to overcome resistance.

Yeast Short-Lived Actin-Associated Protein Forms a Metastable Prion in Response to Thermal Stress.

Self-perpetuating ordered protein aggregates (amyloids and prions) are associated with a variety of neurodegenerative disorders. Although environmental agents have been linked to certain amyloid diseases, the molecular basis of their action remains unclear. We have employed endogenous yeast prions as a model system to study environmental control of amyloid formation. A short-lived actin-associated yeast protein Lsb2 can trigger prion formation by other proteins in a mode regulated by the cytoskeleton and ubiquitin-dependent processes. Here, we show that such a heterologous prion induction is due to the ability of Lsb2 to form a transient prion state, generated in response to thermal stress. Evolutionary acquisition of prion-inducing activity by Lsb2 is traced to a single amino acid change, coinciding with the acquisition of thermotolerance in the Saccharomyces yeast lineage. This raises the intriguing possibility that the transient prion formation could aid in functioning of Lsb2 at higher temperatures.

An extra copy of p53 suppresses development of spontaneous Kras-driven but not radiation-induced cancer.

The tumor suppressor p53 blocks tumor progression in multiple tumor types. Radiation-induced cancer following exposure to radiation therapy or space travel may also be regulated by p53 because p53 has been proposed to respond to DNA damage to suppress tumorigenesis. Here, we investigate the role of p53 in lung carcinogenesis and lymphomagenesis in LA-1 KrasG12D mice with wild-type p53 or an extra copy of p53 (super p53) exposed to fractionated total body irradiation with low linear energy transfer (low-LET) X-rays or high-LET iron ions and compared tumor formation in these mice with unirradiated controls. We found that an additional copy of p53 suppressed both Kras-driven lung tumor and lymphoma development in the absence of radiation. However, an additional copy of p53 did not affect lymphoma development following low- or high-LET radiation exposure and was unable to suppress radiation-induced expansion of thymocytes with mutated Kras. Moreover, radiation exposure increased lung tumor size in super p53 but not wild-type p53 mice. These results demonstrate that although p53 suppresses the development of spontaneous tumors expressing KrasG12D, in the context of exposure to ionizing radiation, an extra copy of p53 does not protect against radiation-induced lymphoma and may promote KrasG12D mutant lung cancer.

Stress-dependent proteolytic processing of the actin assembly protein Lsb1 modulates a yeast prion.

Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI(+)] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI(+)] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI(+)] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.

Prion induction by the short-lived, stress-induced protein Lsb2 is regulated by ubiquitination and association with the actin cytoskeleton.

Yeast prions are self-perpetuating, QN-rich amyloids that control heritable traits and serve as a model for mammalian amyloidoses. De novo prion formation by overproduced prion protein is facilitated by other aggregated QN-rich protein(s) and is influenced by alterations of protein homeostasis. Here we explore the mechanism by which the Las17-binding protein Lsb2 (Pin3) promotes conversion of the translation termination factor Sup35 into its prion form, [PSI(+)]. We show that Lsb2 localizes with some Sup35 aggregates and that Lsb2 is a short-lived protein whose levels are controlled via the ubiquitin-proteasome system and are dramatically increased by stress. Loss of Lsb2 decreases stability of [PSI(+)] after brief heat shock. Mutations interfering with Lsb2 ubiquitination increase prion induction, while a mutation eliminating association of Lsb2 with the actin cytoskeleton blocks its aggregation and prion-inducing ability. These findings directly implicate the UPS and actin cytoskeleton in regulating prions via a stress-inducible QN-rich protein.