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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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BackgroundIn lower-income and middle-income countries, limited research exists on illicit tobacco trade and its responsiveness to taxation. Tobacco taxes are critical in reducing tobacco consumption, thereby improving public health. However, the tobacco industry claims that tax increases will increase illicit tobacco trade. Therefore, research evidence on the size of the illicit cigarette market is needed in Georgia and other low-income and middle-income countries to inform tobacco tax policies.MethodsIn 2017, a household survey using stratified multistage sampling was conducted in Georgia with 2997 smokers, to assess illicit tobacco consumption. Smokers were asked to show available cigarette packs to the surveyors. These were examined for tax stamps and health warnings which allowed for an assessment of illegal cigarette consumption in Georgia.FindingsThe packs shown to surveyors suggest illicit cigarette trade is low (1.5%), although with regional differences, as illicit cigarette packs were present in 6% of the households in Zugdidi. Most illicit cigarettes were purchased at kiosks or informal outlets. This estimate might be conservative, as 28% of respondents did not show any packs to the surveyors.ConclusionsDespite recent tobacco tax increases, illicit cigarette trade in Georgia seems to be negligible. The market is more vulnerable to illicit cigarette trade close to the border with Abkhazia (near Zugdidi). Tighter control or ban of tobacco sales at kiosks and informal outlets may reduce illicit cigarette trade. Further investigation is planned to better understand why a large proportion of survey participants said they had no pack available at home.

RAF protein kinases are effectors of the GTP-bound form of the small guanosine triphosphatase RAS and function by phosphorylating MEK. We showed here that expression of ARAF activated RAS in a kinase-independent manner. Binding of ARAF to RAS displaced the GTPase-activating protein NF1 and antagonized NF1-mediated inhibition of RAS. This reduced ERK-dependent inhibition of RAS and increased RAS-GTP. By this mechanism, ARAF regulated the duration and consequences of RTK-induced RAS activation and supported the RAS output of RTK-dependent tumor cells. In human lung cancers with EGFR mutation, amplification of ARAF was associated with acquired resistance to EGFR inhibitors, which was overcome by combining EGFR inhibitors with an inhibitor of the protein tyrosine phosphatase SHP2 to enhance inhibition of nucleotide exchange and RAS activation.

Background: Peripheral neuropathy occurs in 20-25% of patients with an IgM paraprotein and 40-50% of patients with an IgM monoclonal gammopathy have a detectable anti-myelin associated glycoprotein (MAG) antibody. The presence of an anti-MAG antibody is often associated with a chronic, progressive symmetric demyelinating polyneuropathy that can lead to sensory deficits and gait dysfunction, significantly altering a patient’s quality of life. Prior clinical trials have failed to demonstrate an effective long-term therapy for anti-MAG neuropathy. Additionally, the best manner to assess the evolution of this type of neuropathy has not been elucidated. We designed this clinical trial to evaluate a potential therapy for anti-MAG related neuropathy based on previous reports of subjective improvement in patients with anti-MAG neuropathy treated with rituximab or Bruton’s tyrosine kinase inhibitors. We included multiple neurologic outcome measures so their responsiveness and performance could be assessed in a clinical trial setting. Methods: This is a single-arm open-label phase II trial investigating the use of acalabrutinib and an anti-CD20 monoclonal antibody for the treatment of an IgM-associated anti-MAG mediated neuropathy. Eligible patients must have positive anti-MAG antibody titers with an IgM monoclonal gammopathy of undetermined significance (MGUS) or Waldenström macroglobulinemia (WM). Patients must have a predominantly sensory neuropathy characterized by demyelinating features in nerve conduction studies with a modified Rankin score of ≥1 with progressive symptoms or a modified Rankin score of ≥2. The ECOG performance status must be ≤2. Acceptable organ and marrow function are required. Prior exposure to chemotherapy, Bruton Tyrosine Kinase inhibitors, or other therapies for WM are not permitted except for steroids, IVIg, or an anti-CD20 monoclonal antibody given at least 90 days before study drug initiation. Approximately 33 patients will be enrolled in this study. Patients will be treated with acalabrutinib 100 mg orally twice daily on days 1-28 of each cycle and treatment will be administered until disease progression or unacceptable toxicity. Rituximab (or a biosimilar) will be administered on days 1, 8, 15, and 22 of cycles 1 and 4. The primary endpoint of this trial is to evaluate the overall hematologic response rate (defined as ≥25% reduction in serum IgM) associated with the treatment. The key secondary endpoint is to evaluate the proportion of patients that achieve improvement or stability in neuropathic symptoms based on the Inflammatory Rasch-built Overall Disability Scale (I-RODS) patient-reported disability scale. Additional secondary neurologic endpoints include the INCAT disability score, exam-based outcomes (the MRC distal sum score and INCAT-ISS), measures of function (10-meter walk test, 9-hole peg test), a pain scale, a fatigue scale (FSS), and a neuropathy-specific quality of life scale (IN-QOL). Additional secondary endpoints include time to next treatment, overall survival, hematologic response based on MYD88 and CXCR4 mutational status, change in bone marrow disease burden, and proportion of adverse events associated with treatment. This study is currently open and accruing patients. Clinical trial information: NCT05065554 Sponsor: AstraZeneca Citation Format: Shayna Sarosiek, Christopher T. Doughty, Andrew Branagan, Catherine A. Flynn, Kirsten Meid, Timothy P. White, Megan Little, Carly Leventoff, Steven P. Treon, Jorge J. Castillo. Phase II study of acalabrutinib and an anti-CD20 monoclonal antibody in patients with anti-MAG mediated neuropathy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT550.

Background Small cell lung cancer (SCLC) is an aggressive high grade neuroendocrine tumor (Hann et al. 2019; Bernhardt et al. 2016). Whilst MAPK mutations can be found in roughly 30% of human cancers (Schubbert et al. 2007) including non-small cell lung cancer (NSCLC), genomic and proteomic analyses have indicated suppression of MAPK pathway activity in SCLC (Cerami et al. 2012; Gao et al. 2013). Previous attempts to determine whether this might be therapeutically important (Ravi et al, 1998; Cristea et al. 2016/2020) have had conflicting conclusions. SCLC has recently been defined by the relative expression of four major transcriptional regulators (ASCL1, NEUROD1, POU2F3, YAP1) (Rudin et al., 2019). In this study we aimed to elucidate the effect of MAPK activation in these different SCLC subtypes and explore its therapeutic vulnerability. Results ASCL1-, NEUROD1- and POU2F3- driven SCLC cell lines were transduced with a doxycycline-inducible vector for expression of MEKDDS217D/S221D (MEK1). Activation through MEK1 in ASCL1-driven SCLC cell lines resulted in a significant decrease in cell growth over 9 days. This was associated with G2 cell-cycle arrest and senescence. Expression of MEK1 in the cells of other SCLC subtypes and NSCLC failed to show any appreciable changes in cell growth. Remarkably, athymic mice injected with a MEK1 expressing ASCL1-driven cell line showed significantly slower tumor formation and longer survival than when MEK1 was not expressed. Notably, we observed the opposite when MEK1 expressing NEUROD1-driven cells were injected into athymic mice. We observed strong upregulation of negative feedback regulators DUSP6 and SPRY2 upon MAPK activation which has previously been described in solid tumors and pre-B ALL (Courtois-Cox et al 2006; Shojaee et al. 2015). Interestingly, phosphokinase array demonstrated that, almost exclusively, STAT3 through phosphorylation at S727 was strongly upregulated in the ASCL1-driven subtype after MEK1 expression. This prompted us to look at other regulators of the JAK-STAT pathway and found an increase in phosphorylation of the inhibitory phosphatase PTPN6 (SHP1) in the ASCL1-driven subtype and no STAT5 phosphorylation in any of the subtypes. We next examined whether these cells were sensitive to STAT3 inhibition. Upon treatment with a STAT3 inhibitor, stattic, ASCL1-driven SCLC cells reached their IC50 after 3-5 days in comparison to 9 days for other SCLC subtypes. MEK inhibition through PD0325901 rescued growth inhibition upon MAPK activation and was further associated with a decrease in DUSP6, SPRY2 and pS727 STAT3. JAK1/2 inhibition through ruxolitinib had no effect on cell viability. Summary These findings suggest that ASCL1-driven SCLC in vitro and in vivo is sensitive to activation of MAPK signaling. Our data provides additional understanding of SCLC biology and its complex signaling networks and potential subtype-specific drug susceptibilities. Citation Format: Rebecca Caeser, Christopher Hulton, Emily Costa, Vidushi Durani, Megan Little, Xiaoping Chen, Sam E. Tischfield, Marina Asher, Faruk Erdem Kombak, Shweta S. Chavan, Nisargbhai S. Shah, Metamia Ciampricotti, Elisa de Stanchina, John T. Poirier, Charles M. Rudin, Triparna Sen. STAT3-driven MAPK activation represents a therapeutic vulnerability in ASCL1 high SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 90.

Summary Activation of mitogenic signaling pathways is a common oncogenic driver of many solid tumors including lung cancer. Although activating mutations in the mitogen-activated protein kinase (MAPK) pathway are prevalent in non-small cell lung cancers, MAPK pathway activity, counterintuitively, is relatively suppressed in the more aggressively proliferative small cell lung cancer (SCLC). Here, we elucidate the role of the MAPK pathway and how it interacts with other signaling pathways in SCLC. We find that the most common SCLC subtype, SCLC-A associated with high expression of ASCL1, is selectively sensitive to MAPK activation in vitro and in vivo through induction of cell-cycle arrest and senescence. We show strong upregulation of ERK negative feedback regulators and STAT signaling upon MAPK activation in SCLC-A lines. These findings provide insight into the complexity of signaling networks in SCLC and suggest subtype-specific mitogenic vulnerabilities., Graphical abstract, Highlights • MAPK activation causes cell-cycle arrest and senescence selectively in SCLC-A subtype • MAPK-induced growth inhibition is independent of NOTCH signaling • MAPK activation increases ERK negative feedback and activates STAT3 signaling, Biological sciences; Cell biology; Molecular biology

BackgroundGeorgian illicit cigarette consumption was 1.5% in 2017. In 2018, a new tobacco control law took effect followed by a substantial cigarette excise tax increase in 2019. Research shows these policies reduce tobacco consumption, but the tobacco industry argues they increase illicit trade. There is limited evidence on this, particularly from developing countries.MethodsA panel household survey in Georgia obtained data over three waves: 2017 baseline, 2018 after the tobacco control law took effect and 2019 after taxes increased. A sample of 1578 smokers (and quitters in later waves) from five regions reported their tobacco use and were asked to present a cigarette pack in their possession. These were examined for tax stamps and health warnings to establish legality.FindingsThere was no evidence of an increase in illicit cigarette consumption in Tbilisi, Kutaisi, Akhaltsikhe or Gori in any wave. In Zugdidi, near the Russian-occupied Abkhazia, illicit cigarette consumption was increasing even prior to the tax increase, reaching 30.9% by wave 3. A country-wide shift occurred from manufactured cigarettes to roll-your-own tobacco (whose tax remained unchanged) between waves 2 and 3.ConclusionNo evidence of a country-wide increase in illicit cigarette trade was found after non-fiscal tobacco measures took effect and cigarette taxes increased. Relatively high illicit cigarette consumption in Zugdidi highlights the role of disputed territories and border administration in illicit cigarette supply. Substitution towards roll-your-own tobacco after manufactured cigarette taxes increased demonstrates the importance of equalising taxes on tobacco products to maximise public health benefits.

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

Background Lung cancer is the leading cause of cancer death, killing more people than colon, breast, and prostate cancers combined (Siegel et al. 2013). Small cell lung cancer (SCLC) is a high grade neuroendocrine tumor accounting for ~15% of all lung cancers (Hann et al. 2019). Metastasis is often found at first diagnosis, making SCLC exceptionally lethal (2-year survival &lt5%) (Byers et al. 2015). Whilst MAPK mutations can be found in roughly 30% of human cancers (Schubbert et al. 2007) including non-small cell lung cancer (NSCLC), genomic and proteomic analyses have indicated suppression of MAPK pathway activity in SCLC (Cerami et al. 2012; Gao et al. 2013; Wagle et al. 2018). This striking difference is not well understood and previous attempts to determine whether this might be therapeutically important (Ravi et al, 1998; Cristea et al. 2020) have had conflicting conclusions. SCLC has recently been defined by the relative expression of four major transcriptional regulators (ASCL1, NeuroD1, POU2F3, YAP1) (Rudin et al., 2019). In this study we aimed to elucidate the effect of MAPK activation in these different SCLC subtypes and explore its therapeutic vulnerability. Results We used a doxycycline-inducible vector for expression of MEKDDS217D/S221D (MEK1) in a cohort of ASCL1-, NEUROD1, and POU2F3- driven cell lines. Activation through MEK1 in ASCL1-driven SCLC cell lines resulted in a significant decrease in cell growth over 9 days. This was associated with a decrease in neuroendocrine markers ASCL1 and INSM1, a G2 cell cycle arrest and no significant increase in apoptotic cells. Expression of MEK1 in other SCLC subtypes and NSCLC failed to show any appreciable changes in cell growth. Remarkably, athymic mice injected with a MEK1 expressing ASCL1-driven cell line showed significantly slower tumor formation and longer survival than the ASCL1-driven cell line not expressing MEK1. Previous work established that hyperactivation of BRAFV600E, RAS and MYC can result in oncogene-induced senescence (Serrano et al. 1997) which is caused by upregulation of negative feedback such as SPRY2, DUSP6, ETV5 rather than MAPK pathway activation in some solid tumors and pre-B ALL (Courtois-Cox et al 2006; Shojaee et al. 2015). Similarly, we also observed strong upregulation of DUSP6, SPRY2, but not ETV5 upon MAPK activation. This was especially prominent in ASCL1-driven cell lines that changed from the normal phenotype of being in suspension to a more adherent morphology as a result of MAPK activation. Interestingly, phosphokinase array in the major subtype cell lines after MEK1 activation, demonstrated that, almost exclusively, the STAT pathways, in particular STAT3 through phosphorylation at S727 was strongly upregulated in the ASCL1-driven subtype. This prompted us to examine whether these cells were sensitive to STAT3 inhibition. Upon treatment with a STAT3 inhibitor, Stattic (1μM), ASCL1-driven SCLC cells reached their IC50 after 3-5 days in comparison to 9 days for other SCLC subtypes. NSCLC cells were resistant to STAT3 inhibition. Summary These findings suggest that ASCL1-driven SCLC in vitro and in vivo is sensitive to activation of MAPK signaling in comparison to other SCLC subtypes. Whilst activation of the MAPK pathway might seem counterintuitive to current treatment strategies that aim to inhibit oncogenic signaling, we propose the use of a STAT3 inhibitor that has shown to be effective in vitro. Citation Format: Rebecca Caeser, Christopher Hulton, Vidushi Durani, Emily Costa, Megan Little, Nisargbhai S. Shah, Elisa de Stanchina, John T. Poirier, Charles M. Rudin, Triparna Sen. MAPK pathway activation represents a therapeutic vulnerability in ASCL1-driven SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB186.

Objective The importance of monitoring and deterring the illicit trade of tobacco is illuminated by the recent ratification of the WHO’s Protocol to eliminate illicit trade of Tobacco Products. While the correlatory relationship between tobacco taxation and illicit trade of cigarettes have been measured globally, there is a paucity of research on the impact that a change in tax and price policy measures will have on illicit trade. Measuring this impact objectively is crucial to countering biased estimates aimed at influencing public opinion and policy makers. The objective of the project was to use the exogeneity of tobacco taxation and price policy changes in four low- and middle-income countries (the Gambia, Georgia, Mongolia and South Africa) as natural experiments to measure this impact. Methods Using primary-collected household survey data and the discarded pack collection methodology, we measured illicit trade before and after policy changes. Results In Mongolia, South Africa and Georgia, the policy had no impact on illicit trade. Mongolia observed a decline in their illicit trade during this period, from 15.4% (April 2017) to 6.3% (May/June 2018). In the Gambia, last minute changes to planned policy changes meant that we could only collect one wave of cross-sectional data. However, the data revealed that illicit trade is low at 0.9%-7.3%. We will also relate methodological lessons and recommendations from the studies, specifically recommendations for rapid response research by policymakers on this topic. Conclusions The evidence indicates that tobacco taxation does not have an impact on the illicit trade of cigarettes.

Background. The South African Minister of Health announced in 2016 that he intends to introduce tobacco control legislation that will prohibit smoking in restaurants. This will substantially strengthen the Tobacco Products Control Act (1993, as amended), which currently allows restaurants to have a dedicated, enclosed indoor smoking area. Objectives. To analyse current smoking policies of restaurants, whether and how these policies have changed over the past decade, and restaurateurs’ attitudes to the proposed legislative changes. Methods. From a population of nearly 12 000 restaurants, derived from four websites, we sampled 2 000 restaurants, stratifying by province and type (independent v. chain) and disproportionately sampling small strata to ensure meaningful analysis. We successfully surveyed 741 restaurants, mostly by phone. We also surveyed 60 franchisors from a population of 82 franchisors. Results. Of the restaurants sampled, 44% were 100% smoke-free, 44% had smoking sections outside, 11% had smoking sections inside, and 1% allowed smoking anywhere. Smoking areas were more common in independent restaurants (62%) than franchised restaurants (43%). Of the restaurants with a smoking section, 33% reported that the smoking sections were busier than the non-smoking sections. Twenty-three percent of restaurants had made changes to their smoking policies in the past 10 years, mostly removing or reducing the size of the smoking sections. Customer requests (39%), compliance with the law (35%) and cost and revenue pressures (14%) were the main reasons for changing smoking policies. Of the restaurant respondents 91% supported the current legislation, while 63% supported the proposed legislative changes; 68% of respondents who were aware of the proposed legislation supported it, compared with 58% of respondents who were not aware of the proposed legislation. Conclusions. In contrast to the vehement opposition to the 1999 legislation, which resulted in restaurants going partially smoke-free in 2001, there was limited opposition from restaurants to the proposed legislative changes that would make restaurants 100% smoke-free. Support for the proposed legislation will probably increase as the restaurant industry and the public are made more aware of the proposed legislative changes, although public opinion is vulnerable to tobacco industry-led campaigns.