BACKGROUND:Brigatinib, a selective ALK inhibitor, demonstrated preclinical activity against a range of crizotinib-resistant ALK alterations in NSCLC. We examined associations between brigatinib efficacy and tumor- and plasma-detected driver mutations in crizotinib-resistant ALK fusion-positive NSCLC. PATIENTS AND METHODS:Tumor tissue and plasma circulating tumor DNA (ctDNA) from patients with crizotinib-exposed ALK-positive NSCLC receiving brigatinib in phase 1/2 and phase 2 ALTA trials were analyzed by next-generation sequencing. Objective response rate (ORR) and progression-free survival (PFS) were assessed by mutation status. RESULTS:Ninety-three patients were molecularly profiled at baseline (tumor, 26; ctDNA, 59; 8 with both). Patients received a range of doses, most commonly 90 mg QD. For patients with baseline tumor samples, ORR was 78% (7/9) and median PFS was 11.1 months in patients with secondary ALK mutations co-occurring with ALK fusion, compared with 89% (17/19) and 12.9 months, respectively, in those without ALK mutations. For patients with ctDNA-detectable ALK fusion, ORR was 60% (6/10) and median PFS was 9.2 months in those with secondary ALK mutations, and 50% (10/20) and 21.4 months, respectively, without secondary ALK mutations. The 1 patient with baseline G1202R responded. Six patients had baseline alterations in non-ALK secondary drivers (EGFR, KRAS, NRAS, BRAF, MET); none had response. Emergent G1202R was noted in 3 patients and ALK amplification in 3 patients. CONCLUSION:Brigatinib showed substantial activity in crizotinib-pretreated ALK-positive NSCLC with ALK-dependent mechanisms of resistance. Patients with non-ALK canonical drivers did not respond to brigatinib, suggesting alternative therapeutic approaches in that cohort.

Supplementary Tables (S1-6) and Figures (S1-6)

e23508 Background: Activating mutations in KIT are found in most pts with GIST. First-line imatinib provides initial clinical benefit in advanced disease, but prognosis is poor for pts whose tumors progress, despite 3 other approved tyrosine kinase inhibitors (TKIs). Resistance is typically driven by secondary KIT mutations in the ATP-binding pocket (exons 13/14) or activation loop (exons 17/18). No approved drug has potent activity against both classes of resistance mutations, and many pts have polyclonal resistance after multiple lines of therapy. THE-630 is an investigational pan-variant KIT inhibitor. Based on preclinical models, an average plasma concentration (C av ) of 100 nM is predicted to have potent activity against all major classes of KIT-activating and resistance mutations. Here, we report initial data from a first-in-human study in pts with advanced GIST (NCT05160168). Methods: This ph 1/2 study is assessing the safety (including dose-limiting toxicities [DLTs] and recommended ph 2 dose [RP2D]), pharmacokinetics (PK), and antitumor activity of THE-630. Pts enrolled in the ph 1 portion were ≥18 y, had unresectable or metastatic GIST previously treated with imatinib and ≥1 additional TKI, and had ECOG performance status ≤2. Pts received THE-630 (QD; 28-day cycles) in a 3+3 dose-escalation design. PK was assessed on days 1 and 15 in cycle 1, antitumor activity was assessed by modified RECIST 1.1, and circulating tumor DNA (ctDNA) was assessed using Guardant360. Results: As of 13 Jan 2023, 19 pts had been treated in the ph 1 portion at THE-630 daily doses of 3 mg (n = 3), 4 mg (n = 7), 6 mg (n = 3), 9 mg (n = 3), and 12 mg (n = 3). Median age was 57 y (range 39–72); 13 pts had ≥4 prior TKIs. Median treatment duration was 55 days (range 1–252). PK analysis shows approximately dose-proportional increase in systemic exposure across doses tested. Mean steady-state C av at 12 mg was 47 nM. There was 1 DLT at 4 mg (grade 5 myocardial infarction in a pt with 8 prior TKIs and hyperlipidemia where relationship to THE-630 could not be incontrovertibly ruled out). No other DLTs or related serious adverse events have occurred; 13 pts had treatment-related AEs (TRAEs), 91% of which were grade 1–2. The most common TRAEs were fatigue (n = 4), dry mouth (n = 3), anemia, ALT increase, AST increase, diarrhea, dyspepsia, dyspnea, and hypertension (n = 2 each). Reductions in select KIT-mutant allele fractions in ctDNA were observed, consistent with THE-630 levels achieved thus far. Stable disease (SD) was the best response in 1/7 pts at 4 mg, 1/3 pts at 6 mg, and 3/3 pts at 9 mg. Post data cut, SD was seen in 3/3 pts at 12 mg. Dose escalation continues to determine the RP2D. Conclusions: THE-630 had an acceptable initial safety profile, and escalation continues, as target exposure for pan-variant KIT activity is not yet reached. Reduction in KIT-mutant allele fractions in ctDNA and initial evidence of disease control were observed. Clinical trial information: NCT05160168 .

GSEA analysis of mobocertinib acquired resistant tumors versus response tumors Genes involved in "G2M CHECKPOINT", "MITOTIC SPINDLE" and "MTORC1 SIGNALING" pathways of HALLMARK gene sets.

Supplementary methods, patient information and figure legends

Background: EGFR activating mutations are observed in 10-50% of NSCLC patients. Although the common mutations (L858R [L] and exon 19 deletions [D]) are initially sensitive to first-, second-, and third-generation EGFR inhibitors (e.g., erlotinib [1G], afatinib [2G], and osimertinib [3G], respectively), on-target resistance is observed in a substantial percentage of patients, with T790M (T) and C797S (C) observed most frequently (post-1G/2G and post-3G, respectively). Furthermore, EGFR mutational heterogeneity can increase during treatment with existing inhibitors, inhibition of wild type (WT) EGFR leads to dose-limiting toxicities, and tumors commonly metastasize to the brain. We have designed THE-349 to be a fourth-generation EGFR inhibitor with potent activity against all 8 major single- (L, D), double- (LT, DT, LC, DC), and triple- (LTC, DTC) mutant variants, selectivity over WT EGFR, and activity in the CNS. Methods: Kinome selectivity was evaluated biochemically against a panel of 412 kinases. In vitro potency was determined by assessing effects (IC50s) on human and BaF3 cell lines expressing WT or mutant forms of EGFR. Efficacy studies were conducted using engineered BaF3 cell lines, a luciferase-tagged human NSCLC cell line, and a patient-derived xenograft (PDX) model dosed orally, once-daily at levels that did not exceed the maximum tolerated dose (40 mg/kg THE-349 and 25 mg/kg osimertinib). Results:THE-349 potently inhibited the biochemical activity of all major EGFR mutant variants (IC50s <2 nM) with a high degree of kinome selectivity. In engineered BaF3 cells in vitro, THE-349 potently inhibited viability of all 8 major EGFR mutant variants (L, LT, LC, LTC, D, DT, DC, and DTC), with IC50s <6 nM and with ≥10-fold selectivity over WT EGFR. Similarly, THE-349 demonstrated robust anti-tumor activity against the same 8 EGFR variants in vivo, in all cases inducing deep tumor regressions (>80% TR) in mice implanted with engineered BaF3 cells. In a PDX model from an osimertinib-resistant tumor containing D and DTC variants, THE-349 induced deep and sustained regressions (99% TR) through 56 days of dosing, and tumor regrowth that occurred during a subsequent 5-week dosing holiday was reversed (88% TR) upon redosing with THE-349. In an intracranial tumor model using a triple-mutant (LTC) NSCLC cell line, THE-349 significantly prolonged median survival (from 42.5 to 71.5 days), while osimertinib had no effect. THE-349 has a favorable ADME profile and promising safety profile in IND-enabling toxicology studies. Conclusion: THE-349 is a fourth-generation, CNS-active, mutant-selective EGFR inhibitor that has potent, single-agent activity against all major classes of EGFR activating and resistance mutations. A phase 1 clinical trial of THE-349 is planned. Citation Format: Sen Zhang, Sara Nadworny, Wei-Sheng Huang, Adam Talbot, Emily Ye, Narayana Narasimhan, William C Shakespeare, Victor M Rivera. Preclinical characterization of THE-349, a mutant-selective, CNS-active, fourth-generation EGFR inhibitor to overcome T790M- and C797S-mediated resistance in NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B167.

Supplementary Tables. Table S1. In Vitro Kinase IC50 Values (nM) for Native and Mutant Recombinant KIT. Table S2. Summary of Ba/F3 KIT Cell Lines Generated in this Study. Table S3. Summary of IC50 Viability Values in Ba/F3 KIT Cells. Table S4. Summary of KIT Driven Ba/F3 In Vivo Studies.

GSEA analysis of mobocertinib acquired resistant tumors versus response tumors Genes involved in "G2M CHECKPOINT", "MITOTIC SPINDLE" and "MTORC1 SIGNALING" pathways of HALLMARK gene sets.

GSEA analysis of mobocertinib and T-DM1 treated tumors versus mobocertinib-only treated tumors Genes involved in "INTERFERON ALPHA RESPONSE", "INFLAMMATORY RESPONSE", "INTERFERON GAMMA RESPONSE", "IL2 STAT5 SIGNALING", "TNFA SIGNALING VIA NFKB" and "IL6 JAK STAT3 SIGNALING" pathways of HALLMARK gene sets.

Supplementary Figures. Figure S1. Chemical structures of imatinib, sunitinib, regorafenib and ponatinib. Figure S2. Expression and activation of KIT in engineered Ba/F3 cells. Figure S3. Ponatinib inhibits the phosphorylation of exon 11primary activating, and secondary resistant mutant forms of KIT. Figure S4. Secondary mutants reduce ponatinib potency in Ex9 ins and V560D cell lines. Figure S5. Illustration of the optimal fit of ponatinib to KIT. Figure S6. Impact of compound treatment on KIT signaling in GIST-derived cell lines.

Supplementary Figure Legends from Ridaforolimus (AP23573; MK-8669), a Potent mTOR Inhibitor, Has Broad Antitumor Activity and Can Be Optimally Administered Using Intermittent Dosing Regimens

Supplementary Table 1. Brigatinib in vitro activity (IC50s) in a kinase panel (N=289); Supplementary Figure 1. Chemical structures of brigatinib, crizotinib, ceritinib, and alectinib; Supplementary Figure 2. Brigatinib-mediated inhibition of native or mutant ALK-, ROS1-,FLT3-, and EGFR-driven tumor activity in vitro; Supplementary Figure 3. Cellular and in vivo activity of brigatinib in ROS1 harboring models, compared with crizotinib; Supplementary Figure 4. Comparison of IGF-1R and INSR inhibitory activity of brigatinib with NVP-TAE684; Supplementary Figure 5. Inhibition of ALK phosphorylation in ALK+ cellular models by brigatinib, compared with crizotinib; Supplementary Figure 6. Inhibition of ALK phosphorylation and downstream signaling, and tolerability of brigatinib in vivo, compared with crizotinib; Supplementary Figure 7. TKI activity against parental, and native and mutant EML4-ALK driven Ba/F3 cells; Supplementary Figure 8. TKI antitumor activity against native and mutant EML4-ALK tumors, TKI plasma levels, and tolerability, in vivo.

Drug repositioning seeks to leverage existing clinical knowledge to identify alternative clinical settings for approved drugs. However, repositioning efforts fail to demonstrate improved success rates in late-stage clinical trials. Focusing on 11 approved kinase inhibitors that have been evaluated in 139 repositioning hypotheses, we use data mining to characterize the state of clinical repurposing. Then, using a simple experimental correction with human serum proteins in in vitro pharmacodynamic assays, we develop a measurement of a drug's effective exposure. We show that this metric is remarkably predictive of clinical activity for a panel of five kinase inhibitors across 23 drug variant targets in leukemia. We then validate our model's performance in six other kinase inhibitors for two types of solid tumors: non-small cell lung cancer (NSCLC) and gastrointestinal stromal tumors (GISTs). Our approach presents a straightforward strategy to use existing clinical information and experimental systems to decrease the clinical failure rate in drug repurposing studies.

Introduction While 6 tyrosine kinase inhibitors (TKIs) are available for patients with chronic myeloid leukemia (CML) that is newly diagnosed (imatinib, dasatinib, nilotinib, bosutinib) or refractory to prior treatments (asciminib, ponatinib), only ponatinib has pan-BCR-ABL activity, i.e., maintains potent activity against all single resistance mutations, including the difficult to inhibit T315I and E255V mutants. BCR-ABL mutations are the major known mechanism of resistance not only in CML, but also in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL), where use of ponatinib in newly diagnosed patients is yielding especially encouraging results, presumably due to its ability to suppress emergence of resistance mutations. However, toxicities observed with ponatinib often lead to dose reduction or termination that can limit the benefit of its pan-BCR-ABL profile. The suboptimal safety profile of ponatinib is consistent with its relative lack of kinome selectivity, and its potent activity against KDR, FLT3, and FGFRs (unique amongst the 6 approved BCR-ABL TKIs) could potentially contribute to its unique toxicities. We therefore set out to identify a selective, potent pan-BCR-ABL inhibitor. Methods Compound potency was determined by assessing effects on viability of Ba/F3 cell lines expressing BCR-ABL variants (wild type [WT] and 11 key mutant forms) and off-targets of interest. Preliminary assessment of broader kinome selectivity was performed by calculating the percentage of 88 kinases inhibited by >80% by 1 µM compound (S-score (80)). For efficacy studies, mice implanted with engineered Ba/F3 cell lines were dosed orally once daily. Results Ponatinib potently inhibited BCR-ABL WT, T315I, and E255V with IC50s of 2, 4, and 7 nM respectively, and all 9 other BCR-ABL mutants tested were inhibited with IC50s <4 nM. However, ponatinib also potently inhibited the activity of KDR, FLT3, and FGFR1, with IC50s of 5, 2, and 17 nM, respectively, and had a high kinome S-score (80) of 0.30. Cmpd A is a representative member of a series of compounds we have identified that have a pan-BCR-ABL inhibitory profile and substantially improved selectivity over ponatinib. Cmpd A, potently inhibited BCR-ABL WT, T315I, and E255V, with IC50s of 6, 21, and 55 nM respectively, and 8/9 other BCR-ABL mutants tested were inhibited with IC50s ≤13 nM (Y253H IC50 75 nM). But in contrast to ponatinib, Cmpd A had a high degree of selectivity over FLT3 and FGFR1 (IC50s 1898 and 3323 nM), good selectivity over KDR (IC50 420 nM), and a low kinome S-score (80) of 0.08. We next compared the efficacy and tolerability of ponatinib and Cmpd A in an efficacy model driven by E255V, a key clinical resistance mutant that both compounds inhibited less potently than T315I. At 10 mg/kg, ponatinib inhibited tumor growth by 64% compared to vehicle-treated mice and was well tolerated. At 25 mg/kg, while ponatinib induced strong tumor regression (by 82% compared to baseline), it was poorly tolerated (eg, keratinized skin in 4/6 mice). In contrast 120 mg/kg Cmpd A induced strong tumor regression, by 55%, and was well tolerated (eg, 0/6 mice had keratinized skin). Conclusions We have identified a series of next-generation pan-BCR-ABL inhibitors with substantially improved selectivity over ponatinib. In vitro and in vivo characterization of compounds with further improved selectivity profiles, and the potential to be developed for use in patients with refractory CML and newly diagnosed Ph+ ALL, will be presented.

Supplementary Figure 3 from Ridaforolimus (AP23573; MK-8669), a Potent mTOR Inhibitor, Has Broad Antitumor Activity and Can Be Optimally Administered Using Intermittent Dosing Regimens