Next-generation Tyrosine Kinase Inhibitors Research Articles

Current knowledge about FLT3 gene mutations, exploring the isoforms, and protein importance in AML.

Acute myeloid leukaemia (AML) is a complex haematological malignancy characterised by diverse genetic alterations leading to abnormal proliferation of myeloid precursor cells. One of the most significant genetic alterations in AML involves mutations in the FLT3 gene, which plays a critical role in haematopoiesis and haematopoietic homeostasis. This review explores the current understanding of FLT3 gene mutations and isoforms and the importance of the FLT3 protein in AML. FLT3 mutations, including internal tandem duplications (FLT3-ITD) and point mutations in the tyrosine kinase domain (FLT3-TKD), occur in 25-30% in AML and are associated with poor prognosis. FLT3-ITD mutations lead to constitutive activation of the FLT3 signalling pathway, promoting cell survival and proliferation. FLT3-TKD mutations affect the tyrosine kinase domain and affect AML prognosis in various ways. Furthermore, FLT3 isoforms, including shorter variants, contribute to the complexity of FLT3 biology. Additionally, nonpathological polymorphisms in FLT3 are being explored for their potential impact on AML prognosis and treatment response. This review also discusses the development of molecular treatments targeting FLT3, including first-generation and next-generation tyrosine kinase inhibitors, highlighting the challenges of resistance that often arise during therapy. The final chapter describes FLT3 protein domain rearrangements and their relevance to AML pathogenesis.

Repotrectinib: First Approval.

Repotrectinib (AUGTYRO™) is a next-generation, oral, small-molecule kinase inhibitor of proto-oncogene tyrosine-protein kinase ROS1 (ROS1) and tropomyosin receptor tyrosine kinases (TRKs) TRKA, TRKB, and TRKC. It is being developed by Turning Point Therapeutics, a wholly owned subsidiary of Bristol-Myers Squibb (BMS), for the treatment of locally advanced or metastatic solid tumours, including non-small cell lung cancer (NSCLC). Repotrectinib is a next-generation tyrosine kinase inhibitor rationally designed to inhibit ROS1 and TRK fusion, including in the presence of resistance mutations such as solvent-front mutations. In November 2023, repotrectinib received its first approval in the USA for the treatment of adults with locally advanced or metastatic ROS1-positive NSCLC. Repotrectinib is under regulatory review in China and the EU for NSCLC. Clinical studies of repotrectinib are ongoing in several countries in patients with NSCLC and other solid tumours (including primary central nervous system cancer) across both adult and paediatric patient populations. In addition, preclinical investigation of repotrectinib in multiple myeloma is underway in the USA. This article summarizes the milestones in the development of repotrectinib leading to this first approval for the treatment of locally advanced or metastatic ROS1-positive NSCLC.

Safety and Efficacy of TKIs in very Elderly Patients (≥75 Years) with Chronic Myeloid Leukemia.

While the outcomes of chronic phase chronic myeloid leukemia (CP-CML) patients aged over 65 years have been extensively evaluated in real-life experiences, limited data exist for the very elderly population (i.e., aged ≥ 75 years), especially for next-generation tyrosine kinase inhibitors (TKIs). In this retrospective study, we sought to evaluate the safety and efficacy of TKIs in this particular setting of patients. We conducted a retrospective analysis of a multicenter cohort of 123 newly diagnosed CP-CML very elderly patients. The median age at diagnosis was 80 years (range: 75-96). In the first line, 86.1% of patients received imatinib, 7.1% dasatinib, 5.6% nilotinib, and 0.81% received bosutinib. A total of 31 patients (25.2%) switched to second-line therapy, nine patients to a third line, and one patient to a fourth line of therapy. Resistance to treatment was the primary reason for switching therapy in both the first (64.5%) and second lines (77.7%). At diagnosis, reduced doses were administered in 36.5% of patients, in 61.2% in the second line, and in all patients in subsequent lines of therapy. In the first-line setting, 71.9% of patients achieved an early molecular response (EMR, i.e., 3-month BCR::ABL1IS < 10%); at 6, 12, and 24 months, MR3 was reached by 35.7%, 55.7%, and 75.0% of patients, respectively, with 16.6%, 35.7%, and 51.7% achieving a deep molecular response (DMR) at the same time points. Treatment-free remission (TFR) was successfully attempted in 11 patients. During the follow-up period, adverse events (AEs) were observed in 78.8% of patients, including 22 cases of cardiovascular AEs. Toxicity grade ≥ 3 was more commonly observed in patients treated with standard doses of TKIs compared to reduced doses (p = 0.033). Overall, the median follow-up was 46.62 months (range: 1.8-206.2), and 43 patients died due to non-CML-related causes. Three patients died due to disease progression to advanced (n = 1) and blastic (n = 2) phases. The 5-year overall survival (OS) for the entire cohort was 71.9% (95% CI: 0.63-0.81), with no significant difference between the patients treated with standard doses of TKIs compared to those treated with reduced doses (p = 0.35). TKIs appear to be safe and effective even in very elderly CML patients, and dose optimization strategies yield satisfactory molecular responses for adequate disease control with an improved safety profile.

Abstract C148: Type II ROS1 inhibitors and their liability on ROS1 rearranged non-small cell lung cancer with L2086F mutation

Abstract Introduction: Fusion oncoproteins involving the ROS1 receptor tyrosine kinase have been established as validated therapeutic targets in non-small cell lung cancer. FDA-approved ROS1 tyrosine kinase inhibitors (TKIs) such as crizotinib and entrectinib have shown substantial response rates in these patients, leading to improved outcomes. Historically, the solvent-front ROS1 kinase mutation, G2032R, was a recurrent resistance liability to first generation ROS1 TKI. Next-generation TKIs, including NVL-520, repotrectinib, and taletrectinib are active against the ROS1 G2032R and shown activity in both treatment-naïve and ROS1 G2032R acquired resistance setting. We and others showed that ROS1 L2086F a mutation in catalytic-spine 6 within the kinase domain confers resistance to all first and next-generation type I binding mode TKIs. Cabozantinib, a type II ROS1 inhibitor, is the only FDA-approved TKI that has showed clinical activity against ROS1 L2086F. Recent reports indicated that gilteritinib, an FLT3 TKI, exhibits activity against ALK L1256 (homologous to ROS1 L2086F). In this study, we investigated the efficacy of gilteritinib compared to cabozantinib in ROS1 L2086F resistance model systems. Methods: We tested the effectiveness of ROS1 TKI compared to gilteritinib in Ba/F3 cell lines expressing CD74-ROS1 and EZR-ROS1 resistant mutations. Additionally, we quantified the efficacy of cabozantinib and gilteritinib in ROS1 L2086F using soft agar assays. We engineered CRISPR-Cas9 genome-edited CUTO-28 cells expressing endogenous L2086F mutation, and to validate the activity of gilteritinib and cabozantinib against ROS1 L2086F. Results: The ROS1 kinase mutations F2004C, G2032R and L2086F are resistant to entrectinib inhibition. Cabozantinib, consistent with our previous published data, retains inhibitory activity against ROS1 G2032R and ROS1 L2086F, however the ROS1 F2004C mutation imposes cabozantinib-resistance. Gilteritinib, intriguingly, is active with IC50 ≤ 25 nM against ROS1 F2004C and ROS1 L2086F but not ROS1 G2032R. Immunoblotting studies showed on-target inhibition of ROS1 auto-phosphorylation and effector ERK1/2 activation in the case of ROS1 L2086F mutant cells treated with cabozantinib and gilteritinib. Conclusion: Gilteritinib demonstrates inhibitory activity against ROS1 L2086F mutation. Currently, patients with ROS1 L2086F resistance can be treated with cabozantinib as a second-line treatment option. The preclinical data presented herein suggest that gilteritinib could be an additional, potentially viable alternative to target the highly resistant ROS1 L2086F mutation. Additional studies are needed to explore the use of gilteritinib for ROS1 rearranged NSCLC. Citation Format: Rajat Thawani, Clare Keddy, Matteo Repetto, Kristen Jones, Katelyn Nicholson, Zoe Beach, Alexander Drilon, Monika Davare. Type II ROS1 inhibitors and their liability on ROS1 rearranged non-small cell lung cancer with L2086F mutation [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 C148.

Elenestinib, an Investigational, Next Generation KIT D816V Inhibitor, Reduces Mast Cell Burden, Improves Symptoms, and Has a Favorable Safety Profile in Patients with Indolent Systemic Mastocytosis: Analysis of the Harbor Trial

Introduction Indolent systemic mastocytosis (ISM) is a rare clonal mast cell disease driven by the KIT D816V mutation in nearly all patients. Patients with ISM can present with debilitating acute and chronic cutaneous, gastrointestinal, neurocognitive, and systemic symptoms caused by the release of inflammatory mediators from abnormal mast cells. In some patients, symptoms are not adequately controlled with best supportive care (BSC) medications. Elenestinib (BLU-263) is a novel, investigational, oral, next-generation tyrosine kinase inhibitor that potently and selectively inhibits KIT D816V with limited central nervous system penetration and pharmacokinetics (PK) that support once daily dosing. HARBOR (NCT04910685) is a randomized, double-blind, placebo-controlled, phase 2/3 study assessing efficacy and safety of elenestinib in patients with ISM. Methods Eligible adult patients with ISM, confirmed following central review of bone marrow (BM) pathology, clinical, and laboratory findings per World Health Organization criteria and a moderate-to-severe symptom score based on minimum mean total symptom score (TSS) of the ISM-Symptom Assessment Form (ISM-SAF©2018), were stratified by baseline serum tryptase (&amp;lt;20 ng/mL vs ≥20 ng/mL) and randomly assigned 3:1 to elenestinib 25 mg, 50 mg, or 100 mg once daily + BSC (“elenestinib”) or placebo + BSC (“placebo”). The primary objective of Part 1 was to determine the recommended dose based on safety, PK, and pharmacodynamics. Secondary endpoints included changes in biomarkers of disease burden (serum tryptase, KIT D816V variant allele fraction [VAF], and BM mast cells) and ISM-SAF TSS. Three additional open-label PK groups enrolled patients at 50 mg, 75 mg, and 100 mg to further characterize PK and safety. Results As of data cutoff on July 3, 2023, a total of 122 patients with ISM have received their assigned treatment in Part 1 of the study; 39 were blinded and randomized to elenestinib or placebo, and 83 were treated with open-label elenestinib in the PK groups (21 patients at 50 mg, 34 patients at 75 mg, and 28 patients at 100 mg). Baseline demographics were similar to those reported for the general ISM population. After 12 weeks of therapy, symptom improvement was observed for all dose cohorts, and symptom reduction by TSS was greater for patients on elenestinib versus placebo in the blinded portion of Part 1. Patients receiving elenestinib at 25 mg, 50 mg, and 100 mg doses showed reduction (mean percent) from baseline for tryptase (-15.4%, -50.9%, and -68.4% vs 3.3%, respectively) and KIT D816VVAF (-37.5%, -70.3%, and -77.0% vs -2.5%, respectively) as compared to placebo. Similar reductions in TSS and disease-related biomarkers were observed in the open-label PK cohorts. After a median treatment duration of 35.3 weeks, elenestinib was well-tolerated at all dose levels. There were no treatment-related serious adverse events and no treatment-related adverse events that led to drug discontinuation. Conclusion Elenestinib at all tested doses demonstrated beneficial effects on disease-related symptoms and biomarkers of mast cell burden in this large and maturing cohort of patients with ISM with a moderate-to-severe symptom burden. Elenestinib was well tolerated across all dose levels with a promising early benefit-risk profile in ISM. These preliminary findings will inform dosing for the planned Part 2 of HARBOR, which will examine elenestinib versus placebo in a randomized, blinded, placebo-controlled setting.

Resistance of Lung Cancer to EGFR-Specific Kinase Inhibitors: Activation of Bypass Pathways and Endogenous Mutators.

Epidermal growth factor receptor (EGFR)-specific tyrosine kinase inhibitors (TKIs) have changed the landscape of lung cancer therapy. For patients who are treated with the new TKIs, the current median survival exceeds 3 years, substantially better than the average 20 month survival rate only a decade ago. Unfortunately, despite initial efficacy, nearly all treated patients evolve drug resistance due to the emergence of either new mutations or rewired signaling pathways that engage other receptor tyrosine kinases (RTKs), such as MET, HER3 and AXL. Apparently, the emergence of mutations is preceded by a phase of epigenetic alterations that finely regulate the cell cycle, bias a mesenchymal phenotype and activate antioxidants. Concomitantly, cells that evade TKI-induced apoptosis (i.e., drug-tolerant persister cells) activate an intrinsic mutagenic program reminiscent of the SOS system deployed when bacteria are exposed to antibiotics. This mammalian system imbalances the purine-to-pyrimidine ratio, inhibits DNA repair and boosts expression of mutation-prone DNA polymerases. Thus, the net outcome of the SOS response is a greater probability to evolve new mutations. Deeper understanding of the persister-to-resister transformation, along with the development of next-generation TKIs, EGFR-specific proteolysis targeting chimeras (PROTACs), as well as bispecific antibodies, will permit delaying the onset of relapses and prolonging survival of patients with EGFR+ lung cancer.

Real-World Outcome Analysis of Patients With Stage IV NSCLC Treated With Tyrosine Kinase and Immune Checkpoint Inhibitors

IntroductionOnly a few reports have determined whether recently advanced anticancer drugs, particularly next-generation tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs), prolong the survival of patients with NSCLC in the real world. MethodsTo evaluate the association between recently advanced drugs and patient survival, survival data of 2078 patients with stage IV NSCLC from 1995 to 2022 were analyzed in the present study. The patients were classified into the following six groups in terms of the date of diagnosis: period A, 1995 to 1999; period B, 2000 to 2004; period C, 2005 to 2009; period D, 2010 to 2014; period E, 2015 to 2019; and period F, 2000 to 2022. They were further grouped in terms of EGFR mutation and ALK fusion. ResultsThe median overall survival (mOS) times were 8.9, 11.0, 13.6, 17.9, and 25.2 months in periods A to E, respectively, and the mOS time was not reached in period F. This time was significantly longer in period E than in period D (25.2 versus 17.9 mo, p < 0.005). Moreover, the mOS times of patients with EGFR mutation, those with ALK fusion, and those without both alterations were significantly longer in period E than in period D (46.0 versus 32.0 mo, p < 0.005; not reached versus 36.2 mo, p = 0.018; 14.6 versus 11.7 mo, p < 0.005). The history of treatment with next-generation TKIs and ICIs was found to be associated with overall survival. ConclusionsThe survival of patients with NSCLC was improved from period D to period E, regardless of the presence of driver gene alteration. We found that next-generation TKIs and ICIs may be associated with improvements in overall survival.

Targeted Therapy for Metastatic Non–Small Cell Lung Cancer

The use of next-generation tyrosine kinase inhibitors has led to improved progression-free survival for patients with metastatic non–small cell lung cancer (NSCLC) and those with EGFR-mutant and ALK-positive tumors. Newer therapeutics can now target KRAS G12C mutations, EGFR exon 20 insertions, and ERBB2 (HER2) mutations. Patients with metastatic NSCLC should undergo molecular testing for these mutations as well as for BRAF mutations; MET exon 14 skipping alterations; and ROS1, RET, and NTRK gene rearrangements (fusions). Novel targeted therapeutics are emerging at a fast pace.

Abstract 3400: TY-2136b, a next generation ROS1/TRK/ALK inhibitor, potently inhibits kinase and cell proliferation activities of tumor cells bearing drug-dependent acquired mutations

Abstract Although tyrosine kinase inhibitors (TKIs) with activity against ALK, ROS1, or TRKA-C have significantly improved clinical benefits in patients with diverse tumors harboring ALK, ROS1, or NTRKs rearrangements, drug resistance will be developed and subsequent therapy overcoming acquired resistance remains limited. The resistance is mainly caused by the adaptive mutations evolved in the structural region of ROS1/TRK/ALK kinases, especially solvent front substitutions such as ROS1 G2032R/TRKA G595R/ALK G1202R mutations. Next-generation TKIs targeting these mutations could potentially address this unmet medical need. Currently, several inhibitors, including TPX-0005, are under development in phase I/II clinical trials. Here, we report the finding of a novel small molecule, TY-2136b, which has been identified through a systemic approach against acquired ROS1/TRK/ALK mutations. Kinase assay results suggest that TY-2136b grants similar potency to TPX-0005 inhibiting ROS1 G2032R mutant activity (IC50 1.6 nM vs 2.4 nM ), confers significantly stronger potency than LOXO-101 inhibiting TRKA activity with G595R substitution (IC50 0.8 mM vs 460.1 nM ). The cell proliferation assay results with Ba/F3 cells suggest that TY-2136b is similar potent as TPX-0005 inhibiting cell proliferation of the Ba/F3 strain expressing mutant ROS1 bearing G2032R mutation, a major resistance mutation. TY-2136b also shows potent inhibition towards ROS1, ERK and AKT phosphorylation and downstream signaling in Ba/F3-CD74-ROS1-G2032R cells. Meanwhile, TY-2136b, TPX-0005, and TRK-selective second-generation LOXO-195 inhibitors had similar activity against TRKA G595R and TRKC G623R resistance mutations, but TY-2136b was better than TPX-0005 and LOXO-195 against TRKA G595R/F589L dual mutations in vitro. In vivo studies, TY-2136b showed dose-dependent anti-tumor effect at the dose of 5, 10, and 20 mg/kg, bid, in xenograft tumor models carrying ROS1 G2032R and TRKA G595R mutation, and was more effective than Crizotinib and LOXO-101 at testing dose, and showed better efficacy than TPX-0005 in higher dose. Next, in vivo activity of TY-2136b was examined with xenograft model of KM-12 expressing TPM3-NTRK1 fusion proteins. The results show that TY-2136b and TPX-0005 demonstrate more effectiveness than LOXO-195, and the animals tolerated TY-2136b better than TPX-0005. In the ALK-G1202R xenograft model, TY-2136b showed a significant anti-tumor effect in a certain dose-dependent manner. Taken together, our preclinical data demonstrate that TY-2136b can treat cancers caused by ROS1/TRK/ALK mutations and overcome drug resistance due to acquired solvent-front mutations. Currently, TY-2136b is under first-in-human clinical investigations in the US and China. * To Whom Correspondence should be addressed to: Jun Li, Chengshan Niu and Yusheng Wu Citation Format: Chengshan Niu, Apeng Liang, Yuge Dou, Kaige Ji, Meihua Li, Yanchao Zhao, Yan Zhang, Zhongwei Guo, Aishen Gong, Mingyu Jiang, Shaoqing Chen, Xiugui Chen, Jun Li, Yusheng Wu. TY-2136b, a next generation ROS1/TRK/ALK inhibitor, potently inhibits kinase and cell proliferation activities of tumor cells bearing drug-dependent acquired mutations [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 3400.

Chemotherapy with the use of next-generation TKIs based on MRD has the potential to avoid hematopoietic stem cell transplantation in treatment for adults with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) as postremission treatment is recommended for Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) in current guidelines. However, comparisons of later generation tyrosine kinase inhibitors (TKIs) plus chemotherapy with allo-HSCT have yielded similar outcomes. This meta-analysis was performed to evaluate allo-HSCT in first complete remission (CR1) versus chemotherapy for adult Ph+ALL in the TKI era. Pooled assessment of the hematologic and molecular complete response rates was performed after 3-month TKI treatment. Hazard ratios (HRs) were determined for disease-free survival (DFS) and overall survival (OS) benefit with allo-HSCT. The effect of measurable residual disease status on survival benefit was also analyzed. Thirty-nine retrospective and prospective single-arm cohort studies involving 5054 patients were included. Combined HRs indicated that in the general population, allo-HSCT favorably influenced DFS and OS. Achieving complete molecular remission (CMR) within 3months after starting induction was a favorable survival prognostic factor regardless of whether the patient had undergone allo-HSCT. Among the patients with CMR, survival rates in the nontransplant subgroup were comparable with those in the transplant subgroup, with the estimated 5-year OS of 64% versus 58% and 5-year DFS of 58% versus 51%, respectively. The use of next-generation TKIs results in a higher proportion of patients achieving CMR (ponatinib 82% vs. imatinib 53%), while improving survival in nontransplant patients. Our novel findings suggest that combination chemotherapy plus TKIs leads to a comparable survival benefit as with allo-HSCT for MRD-negative (CMR) patients. This study provides novel evidence for allo-HSCT indications for Ph+ALL in CR1 in the TKI era.

Therapeutic Management of Metastatic Clear Cell Renal Cell Carcinoma: A Revolution in Every Decade.

Clear cell renal cell carcinoma (RCC) oncogenesis is mainly driven by VHL gene inactivation, leading to overexpression of vascular endothelial growth factor (VEGF). The use of tyrosine-kinase inhibitors (TKIs) directed against VEGF and its receptor (VEGFR) revolutionised the management of metastatic renal cancer in the 2000s. The more recent development of next-generation TKIs such as cabozantinib or lenvatinib has made it possible to bypass some of the mechanisms of resistance to first-generation anti-VEGFR TKIs. During the decade 2010-2020, the development of immune checkpoint blockade (ICB) therapies revolutionised the management of many solid cancers, including RCC, in first- and subsequent-line settings. Dual ICB or ICB plus anti-VEGFR TKI combinations are now the standard of care for patients with advanced clear cell RCC. To optimise these combination therapies while preserving patient quality of life, escalation and de-escalation strategies are being evaluated in prospective randomised trials, based on patient selection according to their prognosis risk. Finally, new therapeutic approaches, such as targeting hypoxia-inducible factor (HIF) and the development of innovative treatments using antibody-drug conjugates (ADCs), CAR-T cells, or radiopharmaceuticals, are all potential candidates to improve further patient survival.

Multicenter analysis of stereotactic radiosurgery for multiple brain metastases from EGFR and ALK driven non-small cell lung cancer

IntroductionPatients with brain metastases (BrMs) arising from EGFR and ALK driven non-small cell lung cancer (NSCLC) have favorable prognoses and evolving treatment options. We evaluated multicenter outcomes for stereotactic radiosurgery (SRS) to multiple (≥4) BrMs, where randomized data remain limited. MethodsData were collected retrospectively from 5 academic centers on EGFR and ALK NSCLC who received SRS to ≥4 BrMs with their first SRS treatment between 2008 and 2018. Analyzed endpoints included overall survival (OS), freedom from CNS progression (FFCNSP), and freedom from whole-brain radiotherapy (FFWBRT). ResultsEighty-nine patients (50 EGFR, 39 ALK) received a total of 159 SRS treatments to 1,080 BrMs, with a median follow up of 51.3 months. The median number of BrMs treated with SRS treatment-1 was 6 (range 4–26) and median for all treatments was 9 (range 4–47). Sixteen patients (18 %) had received WBRT prior to SRS treatment-1. The median OS was 24.2, 21.2, and 33.2 months for all patients, EGFR, and ALK subsets, respectively. After multivariable adjustment, only receipt of a next-generation tyrosine kinase inhibitor was associated with OS (HR 0.40, p = 0.005). No differences in OS were observed based on number of BrMs treated. The median FFCNSP was 9.4, 11.6, and 7.5 months, for all patients, EGFR, and ALK subsets, respectively. After multivariable adjustment, the number of BrMs (continuous) treated during treatment-1 was the only negative prognostic factor associated with FFCNSP (HR 1.071, p = 0.045). The 5-year FFWBRT was 73.6 %. ConclusionsThis multicenter analysis over a >10-year period demonstrated favorable OS, FFCNSP, and FFWBRT, in patients with EGFR and ALK driven NSCLC receiving SRS to ≥4 BrMs. These data support SRS as an option in the upfront and salvage setting for higher burden CNS disease in this population.

TKIs for ROS1+ Cancers Get High Marks.

Next-generation tyrosine kinase inhibitor candidates are continuing to emerge for patients with tumor types harboring ROS1 fusions. Phase I findings from two trials, TRIDENT-1 and ARROS-1, indicate high response rates with repotrectinib and NVL-520, respectively. The latter may be a potential best-in-class agent, with potent ROS1 selectivity that avoids off-target toxicity.

CML-074 Pathogenesis of Tyrosine Kinase Inhibitor–Associated Vascular Toxicity in Chronic Myeloid Leukemia

Evidence has emerged demonstrating an association between treatment with certain tyrosine kinase inhibitors (TKIs) and vascular adverse events (VAEs) in chronic myeloid leukemia (CML). An increase in VAEs has been documented with ponatinib, nilotinib, and dasatinib but not with imatinib and bosutinib. We used an endothelial cell (HUVEC) model and an in-vivo hindlimb ischemia mouse model to study TKI-associated VAEs. We show that compared with control-treated cells, ponatinib increased HUVEC apoptosis by 97% and inhibited their VEGF-induced migration by 35%. HUVEC tube formation was significantly abrogated by ponatinib as evidenced by an 87% reduction in tube number, 68% reduction in segment length, 50% reduction in junction number, and 55% increase in the occurrence of isolated segments. Ponatinib's suppressive effect on tube formation was partially rescued by VEGFR2 overexpression, a known ponatinib off-target. Dasatinib also dramatically disrupted tube formation as evidenced by a 97% reduction in tube number, 95% reduction in segment length, 88% reduction in junction number, and 300% increase in the occurrence of isolated segments. Nilotinib was associated with reduced cell viability, whereas imatinib and bosutinib did not affect these processes. Furthermore, HUVECs treated with ponatinib, dasatinib, and nilotinib revealed a differential angiogenesis-related gene expression pattern. The expression profile of imatinib- and bosutinib-treated cells was similar to that of control-treated cells. In our in-vivo model, unilateral femoral artery excision was performed to induce ischemia and stimulate new blood vessel formation in TKI-treated mice. Imatinib-treated mice showed a similar recovery pattern to that of control mice as assessed via Doppler imager. Bosutinib- and dasatinib-treated mice showed an initial delayed recovery but reached an overall recovery of 85% within 28 days. In contrast, ponatinib-treated mice reached overall perfusion of 60% around day 7 without further improvement. These results are in line with the clinical VAE profiles of ponatinib, dasatinib, and nilotinib, implicating endothelial toxicity related to these TKIs and supporting a direct effect of these TKIs on vascular cells. However, the in-vivo effect of these TKIs on endothelial pathogenesis requires further investigation. Understanding TKI-induced VAE pathogenesis could contribute to the future development of safer next-generation TKIs and could potentially influence TKI-personalized tailoring for CML patients.

EP08.02-124 Lorlatinib Treatment Related Adverse Events: Single Centre Real-World Experience in ALK and ROS1-driven NSCLC

Lorlatinib is an ALK and ROS1 targeted next-generation tyrosine kinase inhibitor (TKI). Real-world treatment related adverse event (TRAE) data are limited. We reviewed TRAEs and therapeutic efficacy for advanced non-small-cell lung cancer (NSCLC) patients receiving lorlatinib at our centre.

Innovative Approaches to the Management of Acute Lymphoblastic Leukemia Across the Age Spectrum.

Adults compose nearly half of all patients diagnosed with acute lymphoblastic leukemia (ALL) and historically have had poor survival compared with pediatric patients. Recently approved therapies, such as monoclonal antibodies, CAR T-cell constructs, and next-generation tyrosine kinase inhibitors, have improved survival in relapsed and refractory ALL, and studies are now examining incorporating these treatments and others into the upfront setting. In adolescent and young adult patients, use of pediatric-based regimens has already improved survival compared with historical controls, and the addition of monoclonal antibodies, such as inotuzumab ozogamicin and blinatumomab, may further enhance this survival benefit. In older adults, approaches have centered on minimizing conventional chemotherapy to decrease toxicity by incorporating monoclonal antibodies and other novel therapies to increase efficacy. With the addition of tyrosine kinase inhibitors to chemotherapy for patients with Philadelphia chromosome-positive ALL, survival of this once poor-prognosis ALL subtype now approaches or exceeds outcomes of other subtypes of adult ALL. Further refinements in the backbone treatment regimen and optimal consolidation approaches will likely improve survival further. Although allogeneic hematopoietic stem cell transplant was previously routinely used as consolidation for adults with ALL, incorporation of measurable residual disease and other risk stratification strategies has enabled better identification of patients who will benefit from allogeneic hematopoietic stem cell transplant. Ongoing clinical trials investigating these approaches will continue the evolution of treatment approaches for adults with ALL, with further improvement in outcomes anticipated.

Alectinib versus crizotinib in ALK-positive advanced non-small cell lung cancer and comparison of next-generation TKIs after crizotinib failure: Real-world evidence.

Anaplastic lymphoma kinase (ALK) fusion is a prognostic indicator for patients with non-small cell lung cancer (NSCLC) receiving tyrosine kinase inhibitors (TKIs). The real-world data of ALK TKIs remain a major concern. Patients with ALK-positive advanced NSCLC, who received crizotinib or alectinib treatment in first line, were retrospectively reviewed. ALK status was detected using immunohistochemistry (IHC) or next-generation sequencing (NGS). Clinical outcomes have been comprehensively analyzed between TKIs, ALK fusions, EML4-ALK variants, and next-generation TKIs after crizotinib failure. One hundred sixty-eight patients were successively enrolled (crizotinib, n=109; alctinib, n=59). Alectinib showed consistent superiority in progressive-free survival (PFS) over crizotinib (hazard ratio [HR]: 0.43, 95% confidential interval [CI]: 0.24-0.77, p=0.004). Multivariate Cox regression showed chemotherapy (CT) prior to TKIs or synchronous chemotherapy seemed not to improve PFS compared to ALK inhibitors alone (p > 0.05). And, alectinib was superior to crizotinib in prolonging intracranial PFS (HR 0.12, 95% CI: 0.03-0.49, p=0.003). Patients in EML4 group had a better prognosis than those in non-EML4 group after alectinib administration (HR 0.13, 95% CI: 0.03-0.60, p=0.009). TP53 co-mutations were relatively common (34.0%) and associated with adverse outcome in ALK-positive patients (adjusted HR 2.22, 95% CI: 1.00-4.92, p=0.049). After crizotinib failure, 33 patients received a sequential application of next-generation ALK TKIs. Compared to ceritinib and brigatinib, alectinib might have better PFS (p=0.043). Our results revealed alectinib had better PFS and higher intracranial efficacy compared to crizotinib in ALK-positive NSCLC, and might improve PFS by comparison with ceritinib and brigatinib after crizotinib failure.

P45.04 Phase II Study of Brigatinib in ROS1 Positive Non-Small Cell Lung Cancer (NSCLC) Patients Previously Treated with Crizotinib: Barossa Cohort 2

Brigatinib is a next-generation tyrosine kinase inhibitor targeting ALK and ROS1. Crizotinib is the first drug approved for the treatment of ROS1 fusion-positive NSCLC. Standard treatment for crizotinib-resistant ROS1 positive NSCLC is not established. Barossa is a multicenter, phase II basket study of brigatinib in patients with ROS1 positive solid tumors. This study is composed of three cohorts. ROS1 inhibitor-naïve ROS1 positive NSCLC patients were enrolled in the cohort 1, and ROS1 positive NSCLC patients previously treated with crizotinib were enrolled in the cohort 2.

Early and Next-Generation KIT/PDGFRA Kinase Inhibitors and the Future of Treatment for Advanced Gastrointestinal Stromal Tumor.

The majority of gastrointestinal stromal tumors (GIST) harbor an activating mutation in either the KIT or PDGFRA receptor tyrosine kinases. Approval of imatinib, a KIT/PDGFRA tyrosine kinase inhibitor (TKI), meaningfully improved the treatment of advanced GIST. Other TKIs subsequently gained approval: sunitinib as a second-line therapy and regorafenib as a third-line therapy. However, resistance to each agent occurs in almost all patients over time, typically due to secondary kinase mutations. A major limitation of these 3 approved therapies is that they target the inactive conformation of KIT/PDGFRA; thus, their efficacy is blunted against secondary mutations in the kinase activation loop. Neither sunitinib nor regorafenib inhibit the full spectrum of KIT resistance mutations, and resistance is further complicated by extensive clonal heterogeneity, even within single patients. To combat these limitations, next-generation TKIs were developed and clinically tested, leading to 2 new USA FDA drug approvals in 2020. Ripretinib, a broad-spectrum KIT/PDGFRA inhibitor, was recently approved for the treatment of adult patients with advanced GIST who have received prior treatment with 3 or more kinase inhibitors, including imatinib. Avapritinib, a type I kinase inhibitor that targets active conformation, was approved for the treatment of adults with unresectable or metastatic GIST harboring a PDGFRA exon 18 mutation, including PDGFRA D842V mutations. In this review, we will discuss how resistance mutations have driven the need for newer treatment options for GIST and compare the original GIST TKIs with the next-generation KIT/PDGFRA kinase inhibitors, ripretinib and avapritinib, with a focus on their mechanisms of action.

Abstract 1077: Acquired neratinib resistance is associated with acquisition of HER2 and PIK3CA mutations and can be overcome using potent drug combinations in HER2-positive breast cancer models

Abstract The role of HER2 and PIK3CA mutations in anti-HER2 resistance is gaining more importance in HER2-positive (+) breast cancer. We recently reported that acquired resistance to lapatinib (Lap)-containing regimens is mediated by HER2 L755S, which could be overcome using the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (Nrb). However, less is known about the role of L755S in resistance to next-generation TKIs, particularly when co-occurring with PIK3CA mutations. HER2+ BT474 cell models with primary or sequential acquired resistance (R) to Lap (LapR) or Nrb (NrbR) and their parental (P) counterparts were profiled for alterations in signaling and gene expression by RPPA, western blot, and RNA-seq. For drug efficacy studies, change in cell growth was assessed using imaging-based high-throughput system. Proteomic profiling revealed partial restoration of HER2 phosphorylation and downstream signaling in the LapR and NrbR derivatives. RNA-seq analysis showed that the LapR and NrbR models, but not P cells, harbor HER2 L755S mutation. Importantly, the NrbR but not LapR cells also co-acquire a PIK3CA pathogenic mutation. GSEA analysis of RNA-seq data showed significant downregulation of G2/M checkpoint in the R derivatives compared to P cells, suggesting genetic instability. In line with the presence of HER2 and PIK3CA activating mutations and HER pathway reactivation in the R models, GSEA revealed an enrichment of mTORC1 and KRAS signaling in the R cells. Furthermore, enrichment of epithelial mesenchymal transition signature and downregulation of apical surface genes was observed in the R models compared to P cells, suggestive of their aggressive phenotype. Interestingly, the LapR cells remained sensitive to Nrb, though a higher dose (IC50: ~50nM) was required compared to P cells (IC50: ~2nM). The LapR and NrbR cells were cross-resistant to the HER2-selective TKI tucatinib, and trastuzumab. We recently showed that the NrbR cells were either partially or completely sensitive to poziotinib or TDM1, respectively, suggesting their therapeutic promise against HER2- and PIK3CA-mutant tumors. Of note, our studies using small molecule agents targeting HER and its downstream pathway to facilitate treatment of CNS lesions suggest that AKT or mutant PIK3CA inhibitors are effective only when combined with either neratinib or poziotinib, but not tucatinib, findings which we are currently expanding to xenograft-derived organoids. Overall, our findings suggest a complex disease evolution upon resistance to neratinib but indicate their potentially continued efficacy in overcoming resistance through drug combinations. Ongoing integrative omics analysis to determine the genomic and mutational complexity and landscape will uncover additional mechanistic insights and guide the discovery of other actionable targets. Citation Format: Jamunarani Veeraraghavan, Ragini Mistry, Sarmistha Nanda, Sreyashree Bose, Chia Chia Liu, Vidyalakshmi Sethunath, Martin J. Shea, Tamika Mitchell, Meenakshi Anurag, Michael A. Mancini, Irmina Diala, Alshad S. Lalani, Fabio Stossi, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. Acquired neratinib resistance is associated with acquisition of HER2 and PIK3CA mutations and can be overcome using potent drug combinations in HER2-positive breast cancer models [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 1077.