ALK L1196M

The tight binding of TPX-0131 to residues Arg1202, Met1199 and Arg1120 contribute significantly to overcoming lorlatinib resistance in ALKL1196M/G1202R mutant. These research results are expected to offer insights into the mechanism of TPX-0131 in treating ALKG1202R/L1196M-induced NSCLC resistance and optimizing of ALK inhibitors.

ORNi-PCR followed by ddPCR/real-time PCR enables highly sensitive and accurate detection of ALK mutations by liquid biopsy. Although the clinical data are limited, our results show that these methods are potentially useful for identifying ALK-TKI-resistant NSCLC at the early recurrent phase.

Collectively, resistance of ALK-positive NSCLC cells to crizotinib is induced by G1202R and L1196M mutations through activation of the MDM2/MEK/ERK signalling axis, promoting EMT process and metastasis. These findings suggest that the combination of MDM2 inhibitors and crizotinib could be a potential therapeutic strategy.

A series of novel benzimidazole derivatives were designed and synthesised based on the structures of reported oral available ALK inhibitor and HDAC inhibitor, pracinostat...Compound 3b also inhibited various ALK mutants known to be involved in crizotinib resistance, including mutant L1196M (IC50, 4.9 nM)...At 20 mg/kg, 3b inhibited tumour growth by 85% yet had a negligible effect on mean body weight. These results suggest a attracting route for the further research and optimisation of dual ALK/HDAC inhibitors.

V3 status is also associated with specific patterns of clinically important ALK resistance mutations. These tumor-intrinsic features may inform rational selection and optimization of first-line and consolidative therapy.

In cases of ALK-dependent resistance mutations, lorlatinib could be the first choice as it exhibits the broadest coverage of mutations that lead to resistance against second-generation ALK - TKIs, such as G1202R, and L1196M. In cases of no resistance mutations, atezolizumab, bevacizumab and platinum-based chemotherapy could be the alternative treatment option.

Our findings suggest that real-time quantitative monitoring of ALK resistance mutations during the response period could provide a time course of changes while acquiring resistance mutations. This information would be beneficial for designing an appropriate treatment strategy.

In Cohort 1 (n=21), 15 (71%) pts had progressed on alectinib, 5 (24%) on ceritinib and 1 (5%) on ensartinib. In Cohort 2 (n=18), pts had progressed on crizotinib followed by alectinib (n=9, 50%), ceritinib (n=4, 22%) or brigatinib (n=3, 17%); 2 (11%) pts progressed on crizotinib, then ceritinib followed by alectinib...Of 5 pts with ALK resistance mutations (I1171N, V1180L, L1196M, G1202R, and E1210K) who subsequently received lorlatinib, repeat ctDNA NGS after 2 months demonstrated clearance of ALK resistance mutation(s) in 4/5 (80%) pts (i.e. undetectable on repeat testing)... Resistance mechanisms to ALK inhibitors are heterogenous, including both ALK dependent and independent pathways. DDR and cell cycle gene alterations were commonly detected and may represent previously unreported acquired resistance alterations. Comprehensive ctDNA NGS analysis at progression may help detect novel resistance alterations in patients on ALK inhibitors.

The dynamic changes of genomic characteristics during lorlatinib treatment revealed the mutation landscape of Chinese patients with NSCLC after ALK-TKI resistance, indicated that the resistance profile of lorlatinib were heterogeneous, which laid the foundation for subsequent treatment to overcome lorlatinib resistance.

19 novel imidazo[1,2-b]pyridazine macrocyclic derivatives were designed, synthesized, and tested for their biological activities in an effort to develop ALK inhibitors that would overcome second-generation ALK-TKIs, particularly the G1202R mutation and the lorlatinib-resistant L1196M/G1202R double mutations...This was equally effective to the reference drug Repotrectinib (IC = 40, 164, and 208 nM). The kinase selectivity profile, liver microsome stability test and in vivo pharmacokinetic properties in SD rats of compound O-10 were further evaluated. O-10 was regarded as an effective ALK inhibitor for the treatment of mutations overall.

Analysis of two tissue microarrays comprised of 130 tumor cores from multiple tissue sites of the first 10 NSCLC RTD donors revealed that 66% of tumor cores had >1% LAG3 expression in CD3+ T cells. Among the three patients with ALK fusion-positive NSCLC, 83% (5 of 6 cores), 85% (6 of 7 cores), and 100% (18 of 18 cores) of the tissue cores had >1% LAG3 expression in T cells. The median LAG3 expression in T cells measured by mIF was 1.8% in 99 tumor cores from the ALK fusion-negative study cohort versus 7.3% in the 31 cores from the ALK fusion-positive subset (p=0.004).

Second- (ceritinib, alectinib, brigatinib, and ensartinib) and third-generation (lorlatinib) ALK TKIs have higher intracranial responses than crizotinib but are still limited by emergence of resistance mutations, most commonly G1202R-containing single and compound mutations. Patient-derived models are widely viewed as among the most disease-relevant models for evaluating new therapies and drug resistance. Using a xenograft model derived from an alectinib-relapsed patient, we showed that NVL-655 had high intracranial activity against brain tumors bearing the ALK G1202R mutation that confers resistance to multiple ALK TKIs. NVL-655 is being evaluated in a Phase 1/2 clinical trial for patients with advanced NSCLC and other solid tumors harboring ALK rearrangement or activating ALK mutation (ALKOVE-1): NCT05384626.

No abstract available

Despite early progression within 3 mo under crizotinib, a durable response was achieved with alectinib...Another rebiopsy revealed ALK:p.L1196M, but the tumor did not respond to brigatinib or carboplatin/pemetrexed, before stabilization under lorlatinib...Lorlatinib retained efficacy in the heavily pretreated setting, while its upfront use could possibly have prevented the stepwise emergence of compound ALK mutations. Furthermore, the disease course was more aggressive and OS shorter compared to the V2/TP53wt ALK+ lung adenocarcinoma, while crizotinib, ceritinib and brigatinib did not confer the benefit expected according to NGS results, which also underline the need for more potent drugs against ALK in the high-risk setting of neuroendocrine histology.

Five tyrosine kinase inhibitors (TKIs) have been approved for treatment of ALK-positive non-small cell lung cancer (NSCLC), including first- (crizotinib), second- (ceritinib, alectinib, and brigatinib), and third- (lorlatinib) generation therapies. NVL-655 showed activity in diverse patient-derived models of ALK-positive cancers, including ones that exhibited clinical resistance to prior-generation ALK TKIs. The broad preclinical activity of NVL-655 suggests potential utility as a best-in-class therapy for ALK-positive cancer patients.

Of these, 52 received first-line alectinib treatment (1L) and 56 received alectinib after crizotinib resistance (2L). Also, NF2-mutant patients only had a median PFS of 4 months, significantly poorer than those without (HR 11.1, 95CI 2.17-50; p<0.001). Conclusions By analyzing different mutational profiles of ALK-rearranged patients after alectinib treatment, we proposed MET and NF2 as putative mechanisms that conferred resistance to early resistance of first-line alectinib, which shone light on future tailored treatment for ALK-positive NSCLCs.

Results Of the 133 pts who progressed on alectinib, with or without prior crizotinib or chemotherapy, 131 had evaluable baseline plasma data. The presence of secondary ALK mutations at baseline was associated with a similar ORR (34.5%) and median DoR (5.6 months) and shorter median PFS (3.7 months), with response/clinical benefit observed in pts with G1202R, L1196M, and I1171N ALK secondary mutations. Table: 1009P Brigatinib efficacy parameters a according to baseline ALK mutation status conclusions Brigatinib showed clinically meaningful activity in pts with alectinib-resistant ALK+ NSCLC with or without a detectable ALK fusion and/or secondary mutations.

In 20 eligible patients (median [m]age 63 years [range, 40-89], females 75%, adenocarcinoma 100%, failure of alectinib 90%, FoundationOne Liquid CDx 80%), CGP has altered NLST recommendation in 30% of cases (Figure 2)... CGP has a significant impact on the decision-making process in ALK-rearranged aNSCLC following progression on 2nd/3rd-generation ALK TKIs.

Crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib are approved tyrosine kinase inhibitors (TKIs) for ALK-positive NSCLC, but durability of response is partly limited by ALK resistance mutations...Ascites mononuclear cells were isolated by Ficoll centrifugation and cultured in media (DMEM F12 Glutamax, 10% antibiotics/antimycotics, 10% FBS, hydrocortisone, adenine, RockInhibitor, and 1/10 cholera toxin)... NVL-655 has demonstrated activity in many preclinical models bearing ALK compound mutations, including the MR448re (EML4-ALK v3 G1202R/T1151M) model. The potent preclinical activity of NVL-655 suggests potential clinical utility for ALK-positive patients, including those with resistant compound mutations. The MR448re PDC and xenograft are valuable additions to the ALK therapeutic research landscape where there is limited availability of patient-derived models with lorlatinib-resistant ALK compound mutations.

In 20 eligible patients (median [m]age 63 years [range, 40-89], females 75%, adenocarcinoma 100%, failure of alectinib 90%, FoundationOne Liquid CDx 80%), CGP has altered NLST recommendation in 30% of cases...mOS was 13.2 months (95% CI, 2.9-NR) and 13.0 months (95% CI, 6.0-NR) in groups 1 and 2, respectively (p-0.86). CGP has a significant impact on the decision-making process in ALK-rearranged aNSCLC following progression on 2/3-generation ALK TKIs.

Another pt with the G1202R mutation following alectinib treatment had tumor shrinkage of 27.9%. APG-2449 has a favorable safety and PK profile and was well tolerated in 84 subjects. Preliminary efficacy was observed in pts whose disease was resistant to second-generation TKIs, especially among those with brain metastases, and in TKI-naïve pts. Biomarker data indicated potential target engagement on FAK and immunomodulatory effects of APG-2449.

cfDNA-NGS facilitates detection of ALK fusions and resistance mutations, assessment of prognosis, and monitoring dynamic changes of genomic alterations in ALK+ NSCLC treated with ALK-TKI.

Estimated CNS concentrations exceeded the derived CNS C in all patients for wild-type ALK and the ALK L1196M mutation and in 35.8% of patients for the ALK G1202R mutation. Projected lorlatinib CNS concentrations were consistent with the high intracranial response rates reported in clinical trials and provide further evidence of the potent CNS penetration of lorlatinib.

Sequential postprogression plasma profiling revealed that increased lines of ALK inhibitors can accelerate the accumulation of ALK resistance mutations and may lead to treatment-refractory compound ALK mutations. The selection for optimal first-line TKI is very important to achieve a more efficacious long-term strategy and prevent the emergence of on-target resistance, which may provide guidance for clinical decision making.

Herein, we present an acquired TPM3-NTRK1 fusion resistant to larotrectinib in lung adenocarcinoma with EML4-ALK fusion progressed on lorlatinib.Case Presentation: A 48-year-old male with stage IV adenocarcinoma of lung with metastatic disease of bone was initially treated with carboplatin, pemetrexed, and pembrolizumab...Upon progression of disease (PD), systemic therapy regimen was switched to combination of carboplatin, paclitaxel, and bevacizumab...Subsequently, he was started on alectinib 600 mg twice a day given EML 4-ALK fusion mutation [4.8% of variant allele frequency (VAF)] in circulating tumor DNA (ctDNA) NGS assay...We report the case of acquired TPM3-NTRK1 fusion resistant to larotrectinib and acquired ALK L1196M in lung adenocarcinoma with EML4-ALK fusion progressed on lorlatinib. Further investigations are warranted to explore the mechanism of resistance to ALK TKIs.

ALK rearrangements are detected in approximately 4% of patients with advanced non-small cell lung cancer (NSCLC), and the tyrosine kinase inhibitors (TKIs) crizotinib, alectinib, brigatinib, ceritinib, and lorlatinib have been approved by the FDA for the treatment of ALK-positive NSCLC. The dPCR method was highly sensitive in detecting gene mutations with low allele frequency. It was also found to be able to monitor variable resistance mutations over time during ALK-TKI treatment. In addition, we found that the appearance of G1202R did not always indicate clinical acquisition of resistance that required drug modification.

More than 60% of nonsmall cell lung cancer (NSCLC) patients show a positive response to the first ALK inhibitor, crizotinib, which has been used as the standard treatment for newly diagnosed patients with ALK rearrangement...These preclinical data support XMU-MP-5 as a novel selective ALK inhibitor with high potency and selectivity. XMU-MP-5 holds great promise as a new therapeutic against clinically relevant secondary ALK mutations.

One patient who received first-line lorlatinib treatment achieved partial response for >26.5 months...Brigatinib treatment appeared to be effective for a patient who failed crizotinib treatment because of the L1152V/Q1146K mutations, which might be related to increased binding affinity to these mutants...Although HIP1-ALK-rearranged NSCLC appears to initially respond well to ALK-TKIs, crizotinib resistance may be correlated with the AKAP9-BRAF fusion, ALK compound mutations (L1152V/Q1146K), and the ALK L1196M mutation. Larger studies are needed to evaluate the significance of HIP1-ALK-rearranged NSCLC.

Here, we report, for the first time, a novel, sequentially-evolved EML4-ALK variant 3 G1202R/S1206Y double mutation in cis detected in a patient with ALK-positive NSCLC after disease progression on sequential crizotinib, alectinib, and then lorlatinib. Three-dimensional computer modeling of this double mutation and other G1202R-based double mutations with lorlatinib (ALK G1202R/L1196M, ALK G1202R/F1174C, ALK G1202R/l1198F, ALK G1202R/G1269A) were provided to reveal how these double mutations may confer resistance to lorlatinib through diverse steric hindrances in the ALK kinase domain. In addition, we performed a comprehensive literature review on published acquired double or triple ALK mutations that are resistant to lorlatinib from both patient samples and in vitro mutagenesis experiments.

The mechanisms of ALK TKI resistance were heterogeneous; ALK mutations were found in less than one-third of patients. Compound ALK mutations, which may confer lorlatinib resistance, may occur in crizotinib, ceritinib, and alectinib-resistant lung cancers.

We developed a novel one-step polymerase chain reaction-free nanowire (NW)-based plasma cell-free DNA (cfDNA) assay. In this study, we evaluated the clinical utility of this novel method for the diagnosis of EML4-ALK fusion in advanced non-small cell lung cancer (NSCLC). The NW-based assay and FISH test showed high concordance rate in 99 patients with advanced NSCLC. Serial cfDNA assessment demonstrated this method provided early detection of resistance before radiologic progression during crizotinib treatment. Taken together, plasma cfDNA genotyping by the NW-based cfDNA assay may be useful for the rapid diagnosis of ALK fusion, classifying variants, and early detection of resistance.

Since 2011, with the approval of crizotinib and subsequent approval of four additional targeted therapies, ALK inhibitors have become important treatments for a subset of patients with lung cancer...The solvent front mutation (G1202R) and gatekeeper mutation (L1196M) are major resistance mechanisms to the first two generations of inhibitors while patients treated with the third-generation ALK inhibitor lorlatinib often experience progressive disease with multiple mutations on the same allele (mutations in cis, compound mutations)...Following repeat oral administration of TPX-0131 to rats, brain levels of TPX-0131 were ~66% of those observed in plasma. Taken together, preclinical studies show that TPX-0131 is a CNS-penetrant, next-generation ALK inhibitor that has potency against wild-type ALK and a spectrum of acquired resistance mutations, especially the G1202R solvent front mutation and compound mutations, for which there are currently no effective therapies.

Brigatinib showed clinically meaningful efficacy in Japanese patients with ALK+ NSCLC refractory to alectinib (with or without prior crizotinib).

The combination of a genetic analysis and computational simulation model may make a prediction of resistance mechanisms for overcoming ALK-TKI resistance, and the construction of a genomic and simulation fused database is important for the development of personalized medicine in this field.

Legal entity responsible for the study: Sanomics Limited. Funding: Has not received any funding.