See article, p. 1227

  • Repotrectinib is active against ROS1/TRK/ALK solvent-front mutants that confer resistance to other TKIs.

  • Repotrectinib achieved responses in two patients with ETV6–NTRK33G623E - and CD74–ROS1G2032R-positive tumors.

  • The early clinical activity of repotrectinib supports its ongoing clinical investigation.


Tyrosine kinase inhibitors (TKI) targeting ALK, ROS1, or TRKA–C achieve clinical benefit in patients harboring alterations in these oncogenic drivers. However, the clinical activity of these TKIs is limited by the development of resistance, often caused by the acquisition of mutations in the kinase solvent front. To overcome these resistance mutations, Drilon, Ou, and colleagues investigated the use of repotrectinib, a rationally designed, low-molecular-weight, macrocyclic TKI designed to target wild-type and mutant ALK, ROS1, and TRKA–C. In vitro, repotrectinib had activity against the ALKG1202R, ROS1G2032R, ROS1D2033N, TRKAG595R, and TRKCG623R solvent-front mutations as well as various TRK fusions. In vivo, repotrectinib suppressed the growth of tumors harboring solvent-front mutations that confer resistance to other TKIs. These findings indicate that repotrectinib may be beneficial in patients with ALK, ROS, and NTRK1-rearranged tumors, and it is being evaluated in an ongoing phase I/II clinical trial. In a patient with mammary analogue secretory carcinoma harboring the ETV6–NTRK3 fusion, the G623E resistance mutation was detected after progression on prior TKI therapy, and repotrectinib produced a rapid partial response; treatment is ongoing at 17 months. In a second patient with CD74–ROS1G2032R-positive non–small cell lung cancer, repotrectinib achieved a partial response, with treatment ongoing at 12 months. Collectively, these findings provide clinical proof of concept to suggest that repotrectinib may overcome mutations that confer resistance to other TKIs, supporting further clinical investigation.

See article, p. 1237

  • The SHP2 inhibitor SHP099 prevents MEK inhibition–induced reactivation of RAS–ERK signaling.

  • SHP099 enhances MEK inhibitor activity in pancreatic, lung, breast, and ovarian cancer models.

  • Inhibiting SHP2 might prevent adaptive resistance to MEK inhibitors in a variety of tumor types.


The RAS–ERK pathway is frequently dysregulated in cancer and can be targeted by MEK inhibitors. However, despite initial responses, adaptive resistance often develops, resulting in tumor recurrence and progression. It has been proposed that targeting multiple receptor tyrosine kinases (RTK) upstream of the pathway might suppress resistance. To test this hypothesis, Fedele and colleagues combined SHP099, a specific inhibitor of SHP2 (a phosphatase required for RAS–ERK pathway activation), with MEK inhibition. In vitro, SHP099 abrogated adaptive resistance to MEK inhibition in KRAS-mutant pancreatic and lung cancer cell lines, and this effect could be abrogated by SHP2 mutants that fail to bind SHP099, indicating that on-target SHP2 inhibition overcomes MEK inhibitor resistance. SHP099 treatment prevented the compensatory reactivation of RAS–ERK signaling induced by MEK inhibition, with dual inhibition providing the most durable inhibition of ERK signaling. In vivo, SHP099 suppressed the growth of pancreatic and lung cancer xenografts and orthotopic tumors, and the efficacy was further enhanced by combination treatment with the MEK inhibitor trametinib. Triple negative breast cancers (TNBC) and high-grade serous ovarian cancers (HGSC) frequently express wild-type RAS and exhibit compensatory RTK upregulation and adaptive resistance in response to MEK inhibition. Combination therapy with SHP099 plus trametinib also suppressed tumor growth in TNBC and HGSC models. Collectively, these findings suggest that SHP2 inhibition may prevent adaptive resistance in response to MEK inhibition, thereby enhancing the antitumor efficacy. Thus, combination therapies with SHP099 and a MEK inhibitor may be beneficial in a variety of tumor types.

See article, p. 1250

  • In a phase Ib trial, the TLR9 agonist SD-101 achieved responses in combination with anti–PD-1.

  • SD-101 is well tolerated in patients with advanced melanoma in combination with pembrolizumab.

  • TLR9 agonism may induce immune activation to increase the potency of immune checkpoint blockade.


PD-1 inhibitors, including pembrolizumab, provide clinical benefit in patients with advanced melanoma. However, many patients exhibit primary resistance to anti–PD-1 therapy, and responses depend on preexisting T-cell infiltration. Strategies are needed to enhance the efficacy of immune checkpoint blockade. The TLR9 agonist SD-101 stimulates plasmacytoid dendritic cells, triggering the release of IFNα and maturation into efficient antigen-presenting cells to enhance innate and adaptive immunity. In preclinical studies, intratumoral injection of SD-101 has been shown to enhance the effects of systemic anti-PD–1. In a phase Ib trial, Ribas and colleagues evaluated the safety, efficacy, and pharmacodynamics of systemic pembrolizumab in combination with injection of SD-101 into peripheral metastatic lesions. In total, 22 patients with advanced melanoma were treated, 9 of whom were anti–PD-1/PD-L1 naïve and 13 of whom had received prior anti–PD-1/PD-L1 therapy. The overall response rate was 78% among the 9 anti–PD-1 naïve patients and 15% among the 13 patients who had received prior anti–PD-1 therapy. Overall, the addition of SD-101 to pembrolizumab treatment produced minimal additional toxicity, and the combination therapy was well tolerated. Immune expression profiling of tumor biopsies revealed that treatment increased infiltration of CD8+ T cells, natural killer cells, cytotoxic cells, dendritic cells, and B cells. Collectively, these findings indicate that intratumoral SD-101 in combination with pembrolizumab is well tolerated, induces broad immune activation in the tumor microenvironment, and achieves antitumor activity. Thus, SD-101 may enhance the efficacy of anti–PD-1 therapy in patients with melanoma.

See article, p. 1258

  • The TLR9 agonist SD-101 plus low-dose radiation is safe in patients with indolent lymphoma.

  • In situ vaccination with SD-101 plus radiation reduced the tumor burden at nontreated sites.

  • Therapeutic activation of TLR9 may enhance the antitumor immune response in patients with lymphoma.


Therapeutic strategies to activate the immune system are being developed and used to treat patients with cancer, including in situ vaccine approaches. Preclinical studies have suggested that activation of TLR9 can trigger antitumor immunity in the tumor microenvironment, prompting Frank and colleagues to assess the safety and efficacy of the TLR9 agonist SD-101 (administered by intratumoral injection) in combination with low-dose radiation in a phase I/II trial of 29 patients with untreated indolent lymphoma. Objective responses occurred in 8 of 29 patients (28%), including 1 complete and 7 partial responses. Further, 26 of 29 patients (90%) experienced a reduction in overall tumor burden, and 24 patients had tumor reduction at their nontreated sites. Treatment induced increases in CD8+ and CD4+ effector T cells and decreases in T follicular helper and T regulatory cells in the tumor microenvironment. Further, low levels of CD4+ T regulatory cells, proliferating CD8+ T cells, and granzyme B+ CD8+ T cells were associated with improved outcomes. SD-101 was well tolerated, with grade 3 drug-related adverse events occurring in 8 of 29 (28%) patients and no grade 4–5 adverse events. The results of this phase I/II trial indicate that in situ vaccination with SD-101 is safe and induces systemic antitumor responses in combination with low-dose radiation in patients with indolent lymphoma.

See article, p. 1270

  • Integrated tumor evolution modeling and serial ctDNA profiling predict patient relapse.

  • Longitudinal ctDNA analysis forecasted time to relapse in individual patients on cetuximab therapy.

  • ctDNA analysis more accurately assesses RAS pathway activation status than standard diagnostic assays.


Detection of circulating tumor DNA (ctDNA) has been shown to predict cancer recurrence in a qualitative, but not quantitative, manner. To prospectively characterize the molecular markers of response to the anti-EGFR monoclonal antibody cetuximab in patients with wild-type RAS colorectal cancer, Khan and colleagues performed ctDNA profiling of patients during cetuximab therapy to monitor known mechanisms of resistance to cetuximab and identify novel biomarkers of cetuximab response. Biopsies were obtained at baseline, partial response, and progressive disease (PD) from 22 patients with colorectal tumors identified as wild-type RAS by standard diagnostic assays who received cetuximab monotherapy, 21 of whom were anti-EGFR naïve at the time of therapy. RAS pathway alterations were identified in the baseline ctDNA from 11 of the 22 patients and were associated with poor response rate, and 19 patients harbored RAS pathway alterations at PD. ctDNA and tumor biopsies were highly concordant in colorectal tumors, similar to other tumor types, and evolutionary modeling of ctDNA samples and CEA expression were highly predictive of RECIST relapse. Together, these results validate the predictive power of ctDNA sampling in patients with colorectal cancer, prospectively demonstrate that RAS pathway alterations expand during anti-EGFR antibody therapy, which had previously been demonstrated in retrospective clinical and preclinical studies, and reveal that half of patients with colorectal tumors characterized as wild-type RAS by standard assays in fact harbor RAS pathway alterations detected by ctDNA profiling, and thus would not benefit from anti-EGFR antibody therapy.

See article, p. 1286

  • A breast cancer–specific CTC RNA signature quantifies CTCs and intracellular ER signaling.

  • An elevated CTC score is an early predictor of poor clinical outcome in both localized and metastatic disease.

  • In HR+ cases, persistent ER signaling in CTCs predicts short time to progression on second-line endocrine therapy.


Few biomarkers exist to identify responses to neoadjuvant therapy before surgery in localized breast cancer, and the sensitivity and accuracy of existing methods to assess responses to therapy in metastatic breast cancer are limited. Comparison of gene expression data from normal breast tissue, breast cancer, and hematopoietic cells allowed Kwan, Bardia, and colleagues to identify a breast lineage–specific RNA signature that could quantitatively and noninvasively measure presence of circulating tumor cells (CTC) using a droplet digital PCR assay following microfluidic CTC enrichment from whole-blood specimens. Persistent elevation of this CTC score during neoadjuvant therapy was predictive of residual disease at the time of surgical resection in a prospective cohort of women with high-risk localized breast cancer, and in a second prospective cohort of women with advanced metastatic breast cancer, a high baseline CTC score was predictive of shorter overall survival. A subset of genes included in the breast-specific CTC signature distinguished patients with hormone receptor (HR)–positive disease who rapidly progressed on endocrine therapy; this resistance signature was associated with ER signaling, and its persistence suggests failure to suppress ER activity in these patients. Persistent on-treatment ER signaling measured through CTC-derived RNA was not limited to patients with ESR1 mutations, consistent with the existence of additional resistance mechanisms and suggesting that measurement of the CTC score could provide blood-based, noninvasive functional pharmacodynamic monitoring of ER signaling during endocrine therapy.

See article, p. 1300

  • ARQ 531 is more effective than ibrutinib in mouse models of CLL and Richter transformation.

  • ARQ 531 is cytotoxic to CLL cells harboring wild-type and C481S BTK as well as PLCγ2 mutations.

  • ARQ 531 treatment may be a therapeutic strategy to overcome ibrutinib resistance.


Bruton tyrosine kinase (BTK) drives select hematologic malignancies including chronic lymphocytic leukemia (CLL) and has emerged as a therapeutic target. The irreversible BTK inhibitor ibrutinib improves outcomes in patients with CLL, but the benefit is limited by the development of resistance, with patients going on to relapse via Richter transformation to aggressive lymphoma or CLL progression. Reiff and colleagues hypothesized that the reversible BTK inhibitor ARQ 531 might be effective in ibrutinib-naïve and ibrutinib-resistant CLL cells. The crystal structure of BTK in complex with ARQ 531 demonstrated that ARQ 531 does not interact with C481, mutation of which is associated with ibrutinib resistance, suggesting that ARQ 531 may inhibit both wild-type BTK and the C481S mutant. In vitro, ARQ 531 killed CLL cells in a dose-dependent manner and inhibited cell migration. ARQ 531 inhibited both wild-type and C481S BTK and also inhibited activation of SRC family proteins, AKT, and ERK, resulting in increased cytotoxic activity compared with ibrutinib. In vivo, ARQ 531 was superior to ibrutinib in a mouse model of CLL, producing greater antitumor activity and extending survival. Further, ARQ 531 extended survival in a mouse model of Richter transformation. In cells with PLCγ2 mutations, which confer ibrutinib resistance, ARQ 531 suppressed downstream ERK activation, whereas ibrutinib did not. Altogether, these findings suggest that ARQ 531 may be effective in patients with CLL or Richter transformation who have developed resistance to ibrutinib.

See article, p. 1316

  • SCLC initiation in different cell types gives rise to tumors with distinct metastatic programs in mice.

  • Murine SCLC subtypes are defined by unique patterns of chromatin accessibility and gene expression.

  • Differences in cell-of-origin underlie the discrete evolutionary paths of murine SCLC subtypes.


Small cell lung cancer (SCLC) is a heterogeneous and highly aggressive neuroendocrine tumor, and genomic events, including amplification of the transcription factor nuclear factor I B (NFIB), have nominated drivers of SCLC metastatic progression. However, the genetic and epigenetic changes that drive SCLC metastasis and the role of cell-of-origin in SCLC evolution remain incompletely characterized. Using two genetically engineered mouse models of SCLC, Yang and colleagues found that tumors could arise from different cell types in the lung and that metastatic potential was not an inherent property of SCLC cells; rather, metastases could develop along distinct trajectories depending on the tumor cell-of-origin. Tumors initiated by inactivation of tumor suppressors broadly within lung epithelial cells gave rise to NFIBhi metastases characterized by global changes in chromatin accessibility. In contrast, in mouse models of SCLC where the tumors are initiated specifically in mature pulmonary neuroendocrine cells, metastasis occurred in the absence of NFIB upregulation and without widespread chromatin changes. Primary tumors and metastases from the two mouse models also exhibited differences in gene expression profiles during tumor progression, suggesting that these models represent discrete molecular subtypes of SCLC. Spatial and temporal analyses of tumor initiation support the notion that differences in the tumor cell-of-origin define the distinct evolutionary paths of these SCLC subtypes. These findings demonstrate that SCLC can arise from multiple cell types and emphasize the critical role of cell-of-origin in tumor evolution and eventual metastatic progression.

Note:In This Issue is written by Cancer Discovery editorial staff. Readers are encouraged to consult the original articles for full details.