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Supplementary Fig 2

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.Significance:T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627

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Supplementary Table 1

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The phosphoinositide 3–kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer.Significance:Transcriptomic analysis reveals a key role for the PI3K pathway in regulating RNA splicing, uncovering new mechanisms by which PI3K regulates proliferation and metabolism in breast cancer.See related commentary by Claridge and Hopkins, p. 2216

Supplemental Tables 1-4. Table S1. Sequences of sgRNAs and oligonucleotides; Table S2. GSEA pathway enrichment analysis of enriched and dropout genes in MCF7 and T47D cells; Table S3. The top 2.5% (3745) enriched sgRNAs of whole-genome CRISPR/Cas9 sgRNA knockout screen in MCF7 cells; Table S4. The top 2.5% (3745) enriched sgRNAs of whole-genome CRISPR/Cas9 sgRNA knockout screen in T47D cells.

The phosphoinositide 3–kinase (PI3K) pathway regulates proliferation, survival, and metabolism and is frequently activated across human cancers. A comprehensive elucidation of how this signaling pathway controls transcriptional and cotranscriptional processes could provide new insights into the key functions of PI3K signaling in cancer. Here, we undertook a transcriptomic approach to investigate genome-wide gene expression and transcription factor activity changes, as well as splicing and isoform usage dynamics, downstream of PI3K. These analyses uncovered widespread alternatively spliced isoforms linked to proliferation, metabolism, and splicing in PIK3CA-mutant cells, which were reversed by inhibition of PI3Kα. Analysis of paired tumor biopsies from patients with PIK3CA-mutated breast cancer undergoing treatment with PI3Kα inhibitors identified widespread splicing alterations that affect specific isoforms in common with the preclinical models, and these alterations, namely PTK2/FRNK and AFMID isoforms, were validated as functional drivers of cancer cell growth or migration. Mechanistically, isoform-specific splicing factors mediated PI3K-dependent RNA splicing. Treatment with splicing inhibitors rendered breast cancer cells more sensitive to the PI3Kα inhibitor alpelisib, resulting in greater growth inhibition than alpelisib alone. This study provides the first comprehensive analysis of widespread splicing alterations driven by oncogenic PI3K in breast cancer. The atlas of PI3K-mediated splicing programs establishes a key role for the PI3K pathway in regulating splicing, opening new avenues for exploiting PI3K signaling as a therapeutic vulnerability in breast cancer. Significance: Transcriptomic analysis reveals a key role for the PI3K pathway in regulating RNA splicing, uncovering new mechanisms by which PI3K regulates proliferation and metabolism in breast cancer. See related commentary by Claridge and Hopkins, p. 2216

Summarizing the current preclinical and clinical evidence about bystander effect of antibody-drug conjugates (ADCs) in solid tumors.One of the main challenges of treating solid tumors with ADCs is the heterogeneous expression of the target antigen (Ag), which however may be overcome by the so-called bystander killing effect. This unique, but still debated, feature of certain ADCs is represented by the unintentional payload diffusion from Ag-positive tumor cells to adjacent Ag-negative tumor cells. Some pharmacological characteristics, such as a hydrophobic payload or a cleavable linker, seem to play a major role in this effect. Abundant preclinical evidence of the bystander effect has emerged, and the clinical activity of ADCs in tumors with a heterogeneous Ag expression suggests the relevance of this feature. Additional studies are required to investigate if the bystander effect is necessary for achieving a solid activity with ADCs.

Background: Trastuzumab deruxtecan (T-DXd) has been approved for advanced or metastatic breast, gastric and lung cancers. It has been postulated that irreversible tyrosine kinase inhibitors (TKIs) increase HER2 internalization, thus enhancing T-DXd efficacy. Here we report studies evaluating the combination of TKIs with T-DXd in preclinical models of HER2-low disease. Methods: Surface HER2 was measured with flow cytometry quantifying fluorescently labelled trastuzumab on the surface of HER2-low cell lines HCC38 and HCC1171 treated with afatinib, neratinib, lapatinib or tucatinib for 12 h. HER2 receptor dynamics on cell surface was also measured in NCI-N87 (HER2 IHC 3+) and HCC38 at 24, 48, 72 and 96 h post-treatment with afatinib, and recovery was measured up to 72 h after drug washout. In vivo efficacy was established with T-DXd alone and in combination with afatinib or tucatinib in two gastric patient-derived xenograft (PDX) models, HD-2 (HER2 IHC 1+) and FU (HER2 IHC 2+), and one lung PDX model, MEDI-NSCLC-04 (HER2 IHC 1+). Results: In both HER2-low cell lines, treatment with irreversible inhibitors neratinib or afatinib led to enhanced internalization with a greater than 50% surface HER2 reduction, while treatment with the reversible inhibitors lapatinib or tucatinib led to 13-35% higher surface HER2 levels. Washout experiments indicated that receptor recovery at the plasma membrane begins between 4-12h post-washout of afatinib, with complete recovery at about 72h. Tumor shrinkage was significant in HER2-low gastric PDXs when T-DXd was combined with afatinib, but not with tucatinib. In HD-2, T-DXd 10mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 74% and -3% (with respect to T-DXd) tumor growth inhibition (TGI), respectively. In FU, T-DXd 3mg/kg with afatinib 20mg/kg, and T-DXd 3mg/kg with tucatinib 25mg/kg combinations resulted in 80.3% and 24.7% TGI, respectively. Similarly, in MEDI-NSCLC-04 PDX model, benefit was observed in 5mg/kg T-DXd combination with 20mg/kg QD afatinib (95.3% TGI with respect to monotherapy T-DXd), but not with tucatinib (0.37% TGI). Intermittent schedules (QW, MWF) were also tested for afatinib in combination with T-DXd, with the MWF showing 49.2% TGI and QW showing 33.0 %TGI. In tissue samples from MEDI-NSCLC-04 efficacy study, γH2AX was detected by IHC in T-DXd and T-DXd+afatinib combination. In western blots, a decrease in HER2 as well as pHER2 was observed in response to T-DXd and 20mg/kg afatinib treatment, but not with tucatinib. Conclusions: In HER2 low gastric and lung preclinical models, T-DXd combined with irreversible pan-HER inhibitor induced superior antitumor effects compared to single agents or the combination of T-DXd with reversible inhibitors. These data support the rationale to test combination of T-DXd and pan-HER irreversible inhibitors in the clinical setting. Citation Format: Deepa Bhavsar, Laura Kazlauskas, Flavia Michelini, Matt Griffin, Matt Wilson, Fabiola Cecchi, Liz Croydon, Kim Maratea, Elisa de Stanchina, Yelena Y. Janjigian, Maurizio Scaltriti, Theresa Proia, Jerome Mettetal. Combination of T-DXd with the irreversible pan-HER TKI afatinib drives combination benefit in HER2-low gastric and lung tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3999.

Here, we demonstrate a complete clinical response achieved in a patient with HER2+ metastatic pancreatic ductal adenocarcinoma to a coordinated barrage of anti-HER2, personalized vaccine and checkpoint inhibition immunotherapy, radiation, and chemotherapy. Comprehensive organoid profiling with drug sensitivity screening and drug testing suggested a vulnerability to anti-HER2 directed therapy, facilitating personalized treatment selection for our patient, which contributed to her clinical benefit. Immune response monitoring following personalized vaccine, radiation and checkpoint inhibition showed a sustained increase in neoantigen specific T cell response.

Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.

A novel class of small non-coding RNAs called DNA damage response RNAs (DDRNAs) generated at DNA double-strand breaks (DSBs) in a DROSHA- and DICER-dependent manner has been shown to regulate the DNA damage response (DDR). Similar molecules were also reported to guide DNA repair. Here, we show that DDR activation and DNA repair can be pharmacologically boosted by acting on such non-coding RNAs. Cells treated with enoxacin, a compound previously demonstrated to augment DICER activity, show stronger DDR signalling and faster DNA repair upon exposure to ionizing radiations compared to vehicle-only treated cells. Enoxacin stimulates DDRNA production at chromosomal DSBs and at dysfunctional telomeres, which in turn promotes 53BP1 accumulation at damaged sites, therefore in a miRNA-independent manner. Increased 53BP1 occupancy at DNA lesions induced by enoxacin ultimately suppresses homologous recombination, channelling DNA repair towards faster and more accurate non-homologous end-joining, including in post-mitotic primary neurons. Notably, augmented DNA repair stimulated by enoxacin increases the survival also of cancer cells treated with chemotherapeutic agents.

PI3Kα inhibitors have shown clinical activity in PIK3CA-mutated estrogen receptor-positive (ER+) breast cancer patients. Using whole genome CRISPR/Cas9 sgRNA knockout screens, we identified and validated several negative regulators of mTORC1 whose loss confers resistance to PI3Kα inhibition. Among the top candidates were TSC1, TSC2, TBC1D7, AKT1S1, STK11, MARK2, PDE7A, DEPDC5, NPRL2, NPRL3, C12orf66, SZT2 and ITFG2. Loss of these genes invariably results in sustained mTOR signaling under pharmacological inhibition of the PI3K-AKT pathway. Moreover, resistance could be prevented or overcome by mTOR inhibition, confirming the causative role of sustained mTOR activity in limiting the sensitivity to PI3Kα inhibition. Cumulatively, genomic alterations affecting these genes are identified in about 15% of PIK3CA-mutated breast tumors and appear to be mutually exclusive. This study improves our understanding of the role of mTOR signaling restoration in leading to resistance to PI3Kα inhibition and proposes therapeutic strategies to prevent or revert this resistance.

Amplification of and oncogenic mutations in ERBB2, the gene encoding the HER2 receptor tyrosine kinase, promote receptor hyperactivation and tumor growth. Here we demonstrate that HER2 ubiquitination and internalization, rather than its overexpression, are key mechanisms underlying endocytosis and consequent efficacy of the anti-HER2 antibody–drug conjugates (ADC) ado-trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) in lung cancer cell lines and patient-derived xenograft models. These data translated into a 51% response rate in a clinical trial of T-DM1 in 49 patients with ERBB2-amplified or -mutant lung cancers. We show that cotreatment with irreversible pan-HER inhibitors enhances receptor ubiquitination and consequent ADC internalization and efficacy. We also demonstrate that ADC switching to T-DXd, which harbors a different cytotoxic payload, achieves durable responses in a patient with lung cancer and corresponding xenograft model developing resistance to T-DM1. Our findings may help guide future clinical trials and expand the field of ADC as cancer therapy.Significance:T-DM1 is clinically effective in lung cancers with amplification of or mutations in ERBB2. This activity is enhanced by cotreatment with irreversible pan-HER inhibitors, or ADC switching to T-DXd. These results may help address unmet needs of patients with HER2-activated tumors and no approved targeted therapy.See related commentary by Rolfo and Russo, p. 643.This article is highlighted in the In This Issue feature, p. 627

Coding for proteins has been considered the main function of RNA since the "central dogma" of biology was proposed. The discovery of noncoding transcripts shed light on additional roles of RNA, ranging from the support of polypeptide synthesis, to the assembly of subnuclear structures, to gene expression modulation. Cellular RNA has therefore been recognized as a central player in often unanticipated biological processes, including genomic stability. This ever-expanding list of functions inspired us to think of RNA as a "smart" phone, which has replaced the older obsolete "cellular" phone. In this review, we summarize the last two decades of advances in research on the interface between RNA biology and genome stability. We start with an account of the emergence of noncoding RNA, and then we discuss the involvement of RNA in DNA damage signaling and repair, telomere maintenance, and genomic rearrangements. We continue with the depiction of single-molecule RNA detection techniques, and we conclude by illustrating the possibilities of RNA modulation in hopes of creating or improving new therapies. The widespread biological functions of RNA have made this molecule a reoccurring theme in basic and translational research, warranting it the transcendence from classically studied "cellular" RNA to "smart" RNA.

The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11-RAD50-NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA-RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.

Of the many types of DNA damage, DNA double-strand breaks (DSBs) are probably the most deleterious. Mounting evidence points to an intricate relationship between DSBs and transcription. A cell system in which the impact on transcription can be investigated at precisely mapped genomic DSBs is essential to study this relationship. Here in a human cell line, we map genome-wide and at high resolution the DSBs induced by a restriction enzyme, and we characterize their impact on gene expression by four independent approaches by monitoring steady-state RNA levels, rates of RNA synthesis, transcription initiation and RNA polymerase II elongation. We consistently observe transcriptional repression in proximity to DSBs. Downregulation of transcription depends on ATM kinase activity and on the distance from the DSB. Our study couples for the first time, to the best of our knowledge, high-resolution mapping of DSBs with multilayered transcriptomics to dissect the events shaping gene expression after DSB induction at multiple endogenous sites., DNA double strand breaks (DSBs) are among the most deleterious types of damage and there is strong evidence indicating a relationship between breaks and transcription. Here the authors provide a high-resolution, genome-wide map of induced DSBs and observe ATM-dependent transcriptional repression.

Summary The contribution that oxidative damage to DNA and/or RNA makes to the aging process remains undefined. In this study, we used the hMTH1-Tg mouse model to investigate how oxidative damage to nucleic acids affects aging. hMTH1-Tg mice express high levels of the hMTH1 hydrolase that degrades 8-oxodGTP and 8-oxoGTP and excludes 8-oxoguanine from both DNA and RNA. Compared to wild-type animals, hMTH1-overexpressing mice have significantly lower steady-state levels of 8-oxoguanine in both nuclear and mitochondrial DNA of several organs, including the brain. hMTH1 overexpression prevents the age-dependent accumulation of DNA 8-oxoguanine that occurs in wild-type mice. These lower levels of oxidized guanines are associated with increased longevity and hMTH1-Tg animals live significantly longer than their wild-type littermates. Neither lipid oxidation nor overall antioxidant status is significantly affected by hMTH1 overexpression. At the cellular level, neurospheres derived from adult hMTH1-Tg neural progenitor cells display increased proliferative capacity and primary fibroblasts from hMTH1-Tg embryos do not undergo overt senescence in vitro. The significantly lower levels of oxidized DNA/RNA in transgenic animals are associated with behavioral changes. These mice show reduced anxiety and enhanced investigation of environmental and social cues. Longevity conferred by overexpression of a single nucleotide hydrolase in hMTH1-Tg animals is an example of lifespan extension associated with healthy aging. It provides a link between aging and oxidative damage to nucleic acids.

Summary Senescence is thought to be triggered by DNA damage, usually indirectly assessed as activation of the DNA damage response (DDR), but direct surveys of genetic damage are lacking. Here, we mitotically reactivate senescent human fibroblasts to evaluate their cytogenetic damage. We show that replicative senescence is generally characterized by telomeric fusions. However, both telomeric and extratelomeric aberrations are prevented by hTERT, indicating that even non-telomeric damage descends from the lack of telomerase. Compared with replicative senescent cells, oncogene-induced senescent fibroblasts display significantly higher levels of DNA damage, depicting how oncogene activation can catalyze the generation of further, potentially tumorigenic, genetic damage.

Summary The MRE11-RAD50-NBS1 (MRN) complex supports the synthesis of damage-induced long non-coding RNA (dilncRNA) by RNA polymerase II (RNAPII) from DNA double-strand breaks (DSBs) by an unknown mechanism. Here, we show that recombinant human MRN and native RNAPII are sufficient to reconstitute a minimal functional transcriptional apparatus at DSBs. MRN recruits and stabilizes RNAPII at DSBs. Unexpectedly, transcription is promoted independently from MRN nuclease activities. Rather, transcription depends on the ability of MRN to melt DNA ends, as shown by the use of MRN mutants and specific allosteric inhibitors. Single-molecule FRET assays with wild-type and mutant MRN show a tight correlation between the ability to melt DNA ends and to promote transcription. The addition of RPA enhances MRN-mediated transcription, and unpaired DNA ends allow MRN-independent transcription by RNAPII. These results support a model in which MRN generates single-strand DNA ends that favor the initiation of transcription by RNAPII., Graphical Abstract, Highlights • Purified MRE11-RAD50-NBS1 and RNAPII suffice to reconstitute dilncRNA synthesis • dilncRNA synthesis by RNAPII depends on the ability of MRN to melt DNA ends • RPA enhances MRN-dependent dilncRNA synthesis from DNA ends • Unpaired DNA ends allow MRN-independent dilncRNA synthesis, Sharma et al. show that in an in vitro reconstituted system, the DNA damage-sensing complex MRE11-RAD50-NBS1 (MRN) and RNA polymerase II are sufficient to synthesize RNA transcripts from broken DNA ends. MRN supports transcription by melting DNA ends and allowing RNA polymerase II to initiate RNA synthesis.