Anaplastic Lymphoma Kinase Signaling Research Articles

Abstract 4320: Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL)

Abstract The objectives of this study are to evaluate if anaplastic lymphoma kinase (ALK) inhibition activates autophagy in ALK+ ALCL cells and to determine if that activation can be mitigated using autophagy inhibition. ALCL is an aggressive T-cell non-Hodgkin lymphoma that often presents as advanced stage disease. With the addition of crizotinib or brentuximab vedotin to the ALCL99 chemotherapy backbone, the recently reported Children’s Oncology Group ANHL12P1 study demonstrated a 2-year event-free survival for ALCL patients of 79%. Thus, even with multi-agent chemotherapy combined with targeted therapy, improved therapeutic efficacy is needed. Autophagy is activated when mTORC1 is inhibited, and as ALK signaling is known to promote mTORC1 activity, ALK inhibition is hypothesized to activate autophagy. If present, ALK inhibitor-induced autophagy activation would represent a novel therapeutic opportunity targetable with emerging autophagy inhibitors. An ALK+ ALCL cell line, Karpas 299, was treated with drug combinations for 3 hours, lysates prepared, and proteins separated by molecular weight using standard procedures. ALK inhibitor target engagement was assessed by western blot using phospho-ALKY1604 intensity in 9-point dose response curves. Target engagement of autophagy inhibition (ULK-101 directed against ULK1/2 and hydroxychloroquine (HCQ) targeting the lysosome) was assessed by western blot using phospho-ATG14S29 and LC3B-II intensity in 6-point dose response curves. Cell viability was evaluated using a luminescent CellTiter-Glo assay. Karpas299 cells resistant to crizotinib at 50nM (KCrizR50) were generated by gradually increasing crizotinib concentration in the media until cells grew at a rate similar to parental cells. Crizotinib, ceritinib, and lorlatinib all effectively inhibit ALK phosphorylation in Karpas 299 cells with low nanomolar potency. ULK-101 effectively inhibited pATG14S29 at 5 µM and HCQ effectively inhibited autophagic flux, as evidenced by increased LC3B-II intensity, at 20 µM. When treated with the lowest ALK inhibitor dose needed to achieve complete pALK inhibition (IC99), pATG14 intensity increased representing increased autophagic activity. When combined with ALK inhibition, ULK-101 effectively decreased pATG14S29, blocking the ALK inhibitor-induced autophagy activation. Decreased viability in Karpas 299 cells was observed after crizotinib, ceritinib and lorlatinib treatments with nanomolar potency. KCrizR50 cells had a potency higher than parental Karpas 299 cells for all three ALK inhibitors. ALK inhibition activates autophagy in ALCL cells and autophagy activation is effectively mitigated through concurrent autophagy inhibition. ALK inhibitor resistance in Karpas 299 cells will be further evaluated to determine whether autophagy contributes to the resistant phenotype. Citation Format: Katie L. Buelow, Stephanie Celano, Gregory Poole, Katie Martin, Jeffrey MacKeigan, David J. Hoogstra. Interrogation of autophagy inhibition in anaplastic large cell lymphoma (ALCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4320.

Abstract 1657: Microfluidics-based screening platform identifies a novel therapeutic approach to targeting EML4-ALK driven cancers

Abstract To achieve proliferative dysregulation, a hallmark of cancer, tumor cells utilize a variety of mechanisms to maintain constitutive oncogenic signaling in the absence of extracellular cues. One such mechanism occurs in cells expressing the EML4-ALK fusion protein. This fusion, found in about 4% of non-small cell lung cancer (NSCLC), is a result of an inversion within chromosome 2, placing the 3’ end of the ALK (anaplastic lymphoma kinase) gene downstream of the 5’ end of the EML4 (echinoderm microtubule associated protein-like 4) gene. The resulting fusion protein contains the oligomerization domain and additional disordered sequence elements of EML4 upstream of a functional ALK kinase domain. EML4-ALK is a critical driver for these tumors, promoting aberrant ALK-dependent oncogenic signaling. While tumors harboring the EML4-ALK fusion initially respond to ALK tyrosine kinase inhibitors (TKIs), resistance remains a significant clinical challenge requiring new therapeutic approaches. Recently, it has come to be understood that rather than responding through extracellular proliferative cues, the EML4-ALK fusion protein elicits constitutive ALK signaling through the formation of cytoplasmic biomolecular condensates. These condensates are the result of phase separation and have been shown to be causal for disease phenotypes in cell and animal models driven by EML4-ALK, establishing condensate modulation as a novel means by which to treat TKI-resistant tumors. Here, we have developed a novel proprietary microfluidics-based high throughput screening platform (termed PhaseScanTM) to identify small-molecule modulators of EML4-ALK condensates. Using this technology, we identified compounds that disrupt EML4-ALK condensates in vitro and in cells through a novel mechanism, distinct from that of known ALK TKIs, providing utility in both TKI-resistant as well as TKI-naïve tumors. Further profiling of the mechanism of action (MoA) revealed specific modification within the disordered region of EML4, disruption of EML4 oligomerization, and defective ALK signaling in cancer cells. Thus, our PhaseScanTM methodology enabled the identification of novel, functionally active chemical matter that would have otherwise been challenging to identify utilizing any conventional hit identification methodology. Citation Format: Richard C. Centore, Matthew Watson, Julia Doh, Jerome Cattin, Cinzia Sgambato, Amal Alex, Janhavi Sawant, Prathima Radhakrishnan, Jasmine Cornish, Alex Howarth, Nagakumar Bharatham, William E. Arter, Seema Qamar, Kadi L. Saar, Douglas Williamson, Andrew Seeber, Neils Groenewegen, Magdalena Czekalska, Tadas Kartanas, Niklas Ermann, Ahmed Taher, Tuomas Knowles, Shilpi Arora. Microfluidics-based screening platform identifies a novel therapeutic approach to targeting EML4-ALK driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1657.

A Review on Anaplastic Lymphoma Kinase (ALK) Rearrangements and Mutations: Implications for Gastric Carcinogenesis and Target Therapy.

Gastric adenocarcinoma is a complex disease with diverse genetic modifications, including Anaplastic Lymphoma Kinase (ALK) gene changes. The ALK gene is located on chromosome 2p23 and encodes a receptor tyrosine kinase that plays a crucial role in embryonic development and cellular differentiation. ALK alterations can result from gene fusion, mutation, amplification, or overexpression in gastric adenocarcinoma. Fusion occurs when the ALK gene fuses with another gene, resulting in a chimeric protein with constitutive kinase activity and promoting oncogenesis. ALK mutations are less common but can also result in the activation of ALK signaling pathways. Targeted therapies for ALK variations in gastric adenocarcinoma have been developed, including ALK inhibitors that have shown promising results in pre-clinical studies. Future studies are needed to elucidate the ALK role in gastric cancer and to identify predictive biomarkers to improve patient selection for targeted therapy. Overall, ALK alterations are a relevant biomarker for gastric adenocarcinoma treatment and targeted therapies for ALK may improve patients' overall survival.

A199 ALKAL2/ALK AXIS PROMOTE PERSISTENT VISCERAL PAIN

Abstract Background Long-lasting changes in neural pain circuits precipitate the transition from acute to chronic pain in patients living with inflammatory bowel diseases (IBDs). While significant improvement in IBD therapy has been made to reduce inflammation, a large subset of patients continues to suffer throughout quiescent phases of the disease. Peripheral and central mechanisms contribute to the transition from acute to chronic pain during clinical remission. In the search of new treatments, we previously found that targeting the anaplastic lymphoma kinase (ALK) receptor could have therapeutic value for persistent pain conditions. Aims Here we investigated whether ALKAL2 (ALK ligand) /ALK signaling axis regulates chronic visceral pain. Methods We used the post-inflammatory dextran sodium sulfate (DSS) mouse model of colitis as a well-established rodent model of chronic visceral pain, in which animals display increased VHS 5 weeks after DSS administration in the absence of inflammation as described in IBD patients. To assess the role of ALKAL2/ALK signalling in chronic visceral pain, we pharmacologically blocked ALK receptor using clinically available compound Lorlatinib in the postcolitis model. Moreover we genetically depleted ALKAL2 in gut-innervating nociceptors that express the heat sensitive transient receptor potential vanilloid type 1 (TRPV1) channel, using shRNA vectorized in cell type specific Adeno-associated Viruses. Results We showed that ALKAL2 expression was significatively enhanced in lumbosacral (LS) dorsal root ganglia (DRG) sensory neurons in the post-colitis model. In naïve mice, ALKAL2 administration induced visceral hypersensitivity in an ALK dependant manner. In addition, depletion of ALKAL2 in TRPV1 nociceptors or blocking ALK with the clinically available compound Lorlatinib, reversed visceral hypersensitivity in the post-colitis model. Accordingly, phosphorylation of ALK receptor occurring at the LS of the spinal dorsal horn was reduced. Conclusions Overall, these findings provide novel insights into the mechanisms underlying the transition from acute to chronic visceral pain in IBD. Our work could generate a portfolio of clinically available anti-cancer drugs, including Lorlatinib, as novel pain therapeutics. Repurposed ALK inhibitor could facilitate pain management in in individuals with IBD. Funding Agencies CIHR

ALK signaling primes the DNA damage response sensitizing ALK-driven neuroblastoma to therapeutic ATR inhibition.

High-risk neuroblastoma (NB) is a significant clinical challenge. MYCN and Anaplastic Lymphoma Kinase (ALK), which are often involved in high-risk NB, lead to increased replication stress in cancer cells, suggesting therapeutic strategies. We previously identified an ATR (ataxia telangiectasia and Rad3-related)/ALK inhibitor (ATRi/ALKi) combination as such a strategy in two independent genetically modified mouse NB models. Here, we identify an underlying molecular mechanism, in which ALK signaling leads to phosphorylation of ATR and CHK1, supporting an effective DNA damage response. The importance of ALK inhibition is supported by mouse data, in which ATRi monotreatment resulted in a robust initial response, but subsequent relapse, in contrast to a 14-d ALKi/ATRi combination treatment that resulted in a robust and sustained response. Finally, we show that the remarkable response to the 14-d combined ATR/ALK inhibition protocol reflects a robust differentiation response, reprogramming tumor cells to a neuronal/Schwann cell lineage identity. Our results identify an ability of ATR inhibition to promote NB differentiation and underscore the importance of further exploring combined ALK/ATR inhibition in NB, particularly in high-risk patient groups with oncogene-induced replication stress.

Transcriptomic analysis of neural stem and progenitor cells in comparison with glioblastoma stem cells

Introduction. There is currently no effective therapy for the treatment of glioblastoma. This is partly explained by the high degree of intra- and intertumor heterogeneity of GB, the source of which is believed to be glioblastoma stem cells (GSC). The question of the origin of GSC, which is important for improving clinical outcomes, still remains open. It is believed that GSCs can be formed as a result of oncogenic transformation of neural stem and progenitor cells (NSPcs), which have morphological and functional properties similar to them. Despite significant progress in elucidating the nature of GSCs, little is yet known about the specifically expressed genes and transcripts in these cells in comparison with NSPcs. In this regard, it becomes relevant to study the molecular mechanisms of gliomagenesis using model cell systems based on various clones of GSC.Aim. To conduct a comparative transcriptomic analysis of CD133+-NSPCs and CD133+-GSCs to study the molecular genetic differences between the phenotypes of these cells and identify potential targets for therapeutic effects on GSCs.Materials and methods. Used: highly sensitive transcriptomic analysis on high-density microarrays, cellular technologies, modern bioinformatics analysis.Results. Transcriptomic analysis of CD133+-GSCs and CD133+-NSPCs identified 1825 differentially expressed genes. The biological processes and signaling cascades activated in CD133+-GSCs have been established. It was shown that significant transcriptomic aberrations in CD133+-GSC compared to CD133+-NSPC are primarily due to a group of transcripts regulated by the Shh (Sonic hedgehog), mTOR (mammalian target of rapamycin), ALK (anaplastic lymphoma kinase) signaling cascades, transcription factors E2F1, PRC2, HOXA9, MYC, as well as oncogenes ERBB2 and KRAS. Six transcripts (AQP9, TOX15, HOXB2, STEAP3, TREM1, RFC2) highly expressed in CD133+-GSC and closely associated with the survival of patients with glioblastoma, which may be potential targets for therapeutic effects on CD133+-GSC associated with gliomagenesis, which may be potential targets for therapeutic effects on CD133+-GSC, have been identified and annotated.Conclusion. The data obtained indicate a number of significant molecular genetic differences between the two cell phenotypes, which can be used in the development of new therapeutic approaches for the treatment of glioblastoma.

IGF1R Contributes to Cell Proliferation in ALK-Mutated Neuroblastoma with Preference for Activating the PI3K-AKT Signaling Pathway.

Aberrant activation of anaplastic lymphoma kinase (ALK) by activating point mutation or amplification drives 5-12% of neuroblastoma (NB). Previous work has identified the involvement of the insulin-like growth factor 1 receptor (IGF1R) receptor tyrosine kinase (RTK) in a wide range of cancers. We show here that many NB cell lines exhibit IGF1R activity, and that IGF1R inhibition led to decreased cell proliferation to varying degrees in ALK-driven NB cells. Furthermore, combined inhibition of ALK and IGF1R resulted in synergistic anti-proliferation effects, in particular in ALK-mutated NB cells. Mechanistically, both ALK and IGF1R contribute significantly to the activation of downstream PI3K-AKT and RAS-MAPK signaling pathways in ALK-mutated NB cells. However, these two RTKs employ a differential repertoire of adaptor proteins to mediate downstream signaling effects. We show here that ALK signaling led to activation of the RAS-MAPK pathway by preferentially phosphorylating the adaptor proteins GAB1, GAB2, and FRS2, while IGF1R signaling preferentially phosphorylated IRS2, promoting activation of the PI3K-AKT pathway. Together, these findings reveal a potentially important role of the IGF1R RTK in ALK-mutated NB and that co-targeting of ALK and IGF1R may be advantageous in clinical treatment of ALK-mutated NB patients.

Integration of Multi-omic Data in a Molecular Tumor Board Reveals EGFR-Associated ALK-Inhibitor Resistance in a Patient With Inflammatory Myofibroblastic Cancer.

Inflammatory myofibroblastic tumors (IMTs) are intermediate-grade mesenchymal neoplasms commonly characterized by chromosomal rearrangements causing constitutive activation of anaplastic lymphoma kinase (ALK) and/or ALK mutations causing reduced sensitivity to ALK tyrosine kinase inhibitors (TKI). We present a patient with an IMT who initially responded to first-line alectinib, but who later suffered disease relapse and presently survives with moderate residual disease after receiving second-line lorlatinib. Biopsy specimens were analyzed using next generation sequencing (DNA-seq and RNA-seq) and reverse phase protein microarray (RPPA) as part of an institutional Molecular Tumor Board (MTB) study. An EML4-ALK rearrangement and EGFR activation (pEGFRY1068) were present in both the primary and recurrent tumors, while a secondary ALK I1171N mutation was exclusive to the latter. EGFR signaling in the background of a secondary ALK mutation is correlated with reduced ALK TKI sensitivity in vitro, implicating an important mechanism of drug resistance development in this patient. The RPPA results also critically demonstrate that ALK signaling (ALKY1604) was not activated in the recurrent tumor, thereby indicating that standard-of-care use of third- or fourth-line ALK TKI would not likely be efficacious or durable. These results underscore the importance of real-time clinical integration of functional protein drug target activation data with NGS in the MTB setting for improving selection of patient-tailored therapy.

Abstract 3544: ALK signaling activity stabilizes SLC3A2 protein levels in neuroblastoma tumorigenesis

Abstract Background: Neuroblastoma is the most common childhood tumor that grows in the adrenal glands or sympathetic ganglia. Approximately 10% of pediatric neuroblastoma patients harbor mutations in anaplastic lymphoma kinase (ALK). In a previous study, we identified SLC3A2 as a potential interacting partner with ALK using BioID-based in vivo proximity labeling. SLC3A2 is a multifunctional protein that mediates integrin-dependent signaling, acts as a trafficking chaperone for amino acid transporters, and regulates polyamine transport. No previous link to ALK signaling has been reported and the underlying mechanisms and functional consequences of a SLC3A2-ALK interaction in ALK-driven neuroblastoma are unclear. Materials & Methods: The interaction of SLC3A2 and ALK initially detected with BioID-based in vivo proximity labeling was validated by immunoprecipitation. The effect of ALK signaling on SLC3A2 expression and protein stability was evaluated by quantitative PCR and immunoblotting. The functional effect of SLC3A2 on cell viability were investigated by siRNA and inhibitor treatments in both human and mouse ALK-driven neuroblastoma cells. Results: SLC3A2 was confirmed to interact with ALK in both anti-ALK and anti-SLC3A2 antibody co-immunoprecipitations from NB1 cells (ALK-wild-type) and CLB-GE (ALK-F1174V). Moreover, this interaction was decreased by ALK inhibitor (lorlatinib) treatment. Upon ALKAL2 ligand treatment, ALK signaling increased the protein levels of SLC3A2 and this was abrogated by lorlatinib treatment in NB1 cells. Furthermore, lorlatinib treatment suppressed SLC3A2 expression and significantly affected protein stability in neuroblastoma cells with different ALK mutations including CLB-BAR (ALK exon4-11 deletion), CLB-GAR (ALK-R1275Q), and CLB-GE (ALK-F1174V) during cycloheximide chase analysis. Knockdown of SLC3A2 significantly inhibited cell growth and down-regulated the amino acid transporter SLC7A5 (LAT1) expression in CLB-GE cells (ALK-F1174V). While mono treatment of either lorlatinib (ALK TKI) or AMXT-1501 (polyamine transport inhibitor) only showed moderate effects, combinatorial treatment exhibited a synergistic effect on cell growth in ALK-driven primary-cultured mouse neuroblastoma #9883 (Th-MYCN; Alk-F1178S) and #111 (Th-MYCN; Rosa26_Alkal2) cells. Conclusions: SLC3A2 protein stability and its interaction with ALK is dependent on ALK signaling in an ALK-driven neuroblastoma context. The synergistic effect of combined ALK and polyamine transport inhibitors suggests a potential therapeutic option for ALK-driven neuroblastoma. Citation Format: Wei-Yun Lai, Tzu-Po Chuang, Marcus Borenäs, Ruth Palmer, Bengt Hallberg. ALK signaling activity stabilizes SLC3A2 protein levels in neuroblastoma tumorigenesis. [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 3544.

Pharmacological targeting of the receptor ALK inhibits tumorigenicity and overcomes chemoresistance in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease characterized by its metastatic potential and chemoresistance. These traits are partially attributable to the highly tumorigenic pancreatic cancer stem cells (PaCSCs). Interestingly, these cells show unique features in order to sustain their identity and functionality, some of them amenable for therapeutic intervention. Screening of phospho-receptor tyrosine kinases revealed that PaCSCs harbored increased activation of anaplastic lymphoma kinase (ALK). We subsequently demonstrated that oncogenic ALK signaling contributes to tumorigenicity in PDAC patient-derived xenografts (PDXs) by promoting stemness through ligand-dependent activation. Indeed, the ALK ligands midkine (MDK) or pleiotrophin (PTN) increased self-renewal, clonogenicity and CSC frequency in several in vitro local and metastatic PDX models. Conversely, treatment with the clinically-approved ALK inhibitors Crizotinib and Ensartinib decreased PaCSC content and functionality in vitro and in vivo, by inducing cell death. Strikingly, ALK inhibitors sensitized chemoresistant PaCSCs to Gemcitabine, as the most used chemotherapeutic agent for PDAC treatment. Consequently, ALK inhibition delayed tumor relapse after chemotherapy in vivo by effectively decreasing the content of PaCSCs. In summary, our results demonstrate that targeting the MDK/PTN-ALK axis with clinically-approved inhibitors impairs in vivo tumorigenicity and chemoresistance in PDAC suggesting a new treatment approach to improve the long-term survival of PDAC patients.

Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells

Background aimsThe targeting of solid cancers with chimeric antigen receptor (CAR) T cells faces many technological hurdles, including selection of optimal target antigens. Promising pre-clinical and clinical data of CAR T-cell activity have emerged from targeting surface antigens such as GD2 and B7H3 in childhood cancer neuroblastoma. Anaplastic lymphoma kinase (ALK) is expressed in a majority of neuroblastomas at low antigen density but is largely absent from healthy tissues. MethodsTo explore an alternate target antigen for neuroblastoma CAR T-cell therapy, the authors generated and screened a single-chain variable fragment library targeting ALK extracellular domain to make a panel of new anti-ALK CAR T-cell constructs. ResultsA lead novel CAR T-cell construct was capable of specific cytotoxicity against neuroblastoma cells expressing low levels of ALK, but with only weak cytokine and proliferative T-cell responses. To explore strategies for amplifying ALK CAR T cells, the authors generated a co-CAR approach in which T cells received signal 1 from a first-generation ALK construct and signal 2 from anti-B7H3 or GD2 chimeric co-stimulatory receptors. The co-CAR approach successfully demonstrated the ability to avoid targeting single-antigen-positive targets as a strategy for mitigating on-target off-tumor toxicity. ConclusionsThese data provide further proof of concept for ALK as a neuroblastoma CAR T-cell target.

Abstract 2012: A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS)

Abstract Introduction: ALK is a lineage restricted oncoprotein expressed on the cell surface of NBL as wild-type (WT), mutated, or amplified, and recently shown to be overexpressed in FP RMS. Small molecule inhibitors have shown potent efficacy in NBL with ALK mutations or amplifications, but is limited to a minority of the high-risk population and have demonstrated conferred resistance. We have previously shown the potential of an ADC approach for WT ALK-expressing NBL with a chimeric ALK-directed ADC CDX-0125. Methods: A highly specific humanized ALK antibody KTN0239 was developed with site-specific conjugation of an amine-azido linker conjugated to pyrrolobenzodiazepine (PBD) dimers or duocarmycin using click chemistry. ALK surface expression in ALK amplified (NB-1), WT non-amplified (IMR-32), mutant (Felix, SH-SY5Y, Kelly, NB1-643), and WT null (SK-N-AS) cell lines were quantified with flow cytometry using the primary KTN0239 antibody. Internalization kinetics of KTN0239 were measured using Incucyte® Fabflour-pH labeling. In vitro ADC IC50 values and viability rescue were assessed with a CellTiter-Glo® assay with ADC concentrations ranging from 10 fM to 10 nM. Western blot was used to elucidate cytotoxic mechanism of the ADC. Results: ALK surface protein molecule expression was 21,820 in NB-1 cells, 3,708 in Felix, and 1,426 in SK-N-AS cells. KTN0239 was internalized into NB1 cells with a logarithmic trend, with >65% of antibody internalized within 8 hours. NB-1 cell line viability was rescued to >80% when 2.5 nM of KTN0239 was in competition with the PBD-conjugated ADC at the IC50 concentration. The ADC induced activation of PARP, caspase-3, and γH2AX without attenuation of ALK signaling in NB-1 cells. Conclusions: ADCs directed to ALK-expressing cancers are a promising therapeutic approach. The efficacy of the ADCs are currently being tested in NBL and FP RMS mouse models, and will be reported. Mean IC50 values (pM) of ADCs with a drug antibody ratio (DAR) of 6 in NBL cell lines ADC: PBD-conjugated Duocarmycin-conjugated NB-1 14 71 IMR-32 12 741 Felix 51 110 NB-1643 6.6 1,196 Kelly 517 34,700 SH-SY5Y 693 3,024 SK-N-AS 533 1,420 Citation Format: Alberto D. Guerra, Annie Kennedy, Cynthia Adams, Rawan Shraim, Amber K. Weiner, Chuan Chen, Kateryna Krytska, Colleen Larmour, Smita Matkar, Dimiter Dimitrov, Dontcho V. Jelev, Yael P. Mossé. A humanized anaplastic lymphoma kinase (ALK)-directed antibody-drug conjugate (ADC) demonstrates potent cytotoxicity in neuroblastoma (NBL) and fusion positive rhabdomyosarcoma (FP RMS) [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 2012.

Mutations in ALK signaling pathways conferring resistance to ALK inhibitor treatment lead to collateral vulnerabilities in neuroblastoma cells

BackgroundDevelopment of resistance to targeted therapies has tempered initial optimism that precision oncology would improve poor outcomes for cancer patients. Resistance mechanisms, however, can also confer new resistance-specific vulnerabilities, termed collateral sensitivities. Here we investigated anaplastic lymphoma kinase (ALK) inhibitor resistance in neuroblastoma, a childhood cancer frequently affected by activating ALK alterations.MethodsGenome-wide forward genetic CRISPR-Cas9 based screens were performed to identify genes associated with ALK inhibitor resistance in neuroblastoma cell lines. Furthermore, the neuroblastoma cell line NBLW-R was rendered resistant by continuous exposure to ALK inhibitors. Genes identified to be associated with ALK inhibitor resistance were further investigated by generating suitable cell line models. In addition, tumor and liquid biopsy samples of four patients with ALK-mutated neuroblastomas before ALK inhibitor treatment and during tumor progression under treatment were genomically profiled.ResultsBoth genome-wide CRISPR-Cas9-based screens and preclinical spontaneous ALKi resistance models identified NF1 loss and activating NRASQ61K mutations to confer resistance to chemically diverse ALKi. Moreover, human neuroblastomas recurrently developed de novo loss of NF1 and activating RAS mutations after ALKi treatment, leading to therapy resistance. Pathway-specific perturbations confirmed that NF1 loss and activating RAS mutations lead to RAS-MAPK signaling even in the presence of ALKi. Intriguingly, NF1 loss rendered neuroblastoma cells hypersensitive to MEK inhibition.ConclusionsOur results provide a clinically relevant mechanistic model of ALKi resistance in neuroblastoma and highlight new clinically actionable collateral sensitivities in resistant cells.

Targeting ALK Rearrangements in NSCLC: Current State of the Art.

Anaplastic lymphoma kinase (ALK) alterations in non-small cell lung cancer (NSCLC) can be effectively treated with a variety of ALK-targeted drugs. After the approval of the first-generation ALK inhibitor crizotinib which achieved better results in prolonging the progression-free survival (PFS) compared with chemotherapy, a number of next-generation ALK inhibitors have been developed including ceritinib, alectinib, brigatinib, and ensartinib. Recently, a potent, third-generation ALK inhibitor, lorlatinib, has been approved by the Food and Drug Administration (FDA) for the first-line treatment of ALK-positive (ALK+) NSCLC. These drugs have manageable toxicity profiles. Responses to ALK inhibitors are however often not durable, and acquired resistance can occur as on-target or off-target alterations. Studies are underway to explore the mechanisms of resistance and optimal treatment options beyond progression. Efforts have also been undertaken to develop further generations of ALK inhibitors. This review will summarize the current situation of targeting the ALK signaling pathway.

Tyrosine phosphatases regulate resistance to ALK inhibitors in ALK+ anaplastic large cell lymphoma

AbstractAnaplastic large cell lymphomas (ALCLs) frequently carry oncogenic fusions involving the anaplastic lymphoma kinase (ALK) gene. Targeting ALK using tyrosine kinase inhibitors (TKIs) is a therapeutic option in cases relapsed after chemotherapy, but TKI resistance may develop. By applying genomic loss-of-function screens, we identified PTPN1 and PTPN2 phosphatases as consistent top hits driving resistance to ALK TKIs in ALK+ ALCL. Loss of either PTPN1 or PTPN2 induced resistance to ALK TKIs in vitro and in vivo. Mechanistically, we demonstrated that PTPN1 and PTPN2 are phosphatases that bind to and regulate ALK phosphorylation and activity. In turn, oncogenic ALK and STAT3 repress PTPN1 transcription. We found that PTPN1 is also a phosphatase for SHP2, a key mediator of oncogenic ALK signaling. Downstream signaling analysis showed that deletion of PTPN1 or PTPN2 induces resistance to crizotinib by hyperactivating SHP2, the MAPK, and JAK/STAT pathways. RNA sequencing of patient samples that developed resistance to ALK TKIs showed downregulation of PTPN1 and PTPN2 associated with upregulation of SHP2 expression. Combination of crizotinib with a SHP2 inhibitor synergistically inhibited the growth of wild-type or PTPN1/PTPN2 knock-out ALCL, where it reverted TKI resistance. Thus, we identified PTPN1 and PTPN2 as ALK phosphatases that control sensitivity to ALK TKIs in ALCL and demonstrated that a combined blockade of SHP2 potentiates the efficacy of ALK inhibition in TKI-sensitive and -resistant ALK+ ALCL.

Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation.

Variants of the oncogenic EML4‐ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4‐ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4‐ALK V3 but not V1 protein re‐localises to microtubules, an effect recapitulated in a catalytically inactive EML4‐ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild‐type and catalytically inactive EML4‐ALK V3 proteins, but not a Lys‐Glu salt bridge mutant. We propose that EML4‐ALK foci formation occurs as a result of transient association of stable EML4‐ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4‐ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.

Mapping the Phospho-dependent ALK Interactome to Identify Novel Components in ALK Signaling

Protein-protein interactions (PPIs) play essential roles in Anaplastic Lymphoma Kinase (ALK) signaling. Systematic characterization of ALK interactors helps elucidate novel ALK signaling mechanisms and may aid in the identification of novel therapeutics targeting related diseases. In this study, we used the Mammalian Membrane Two-Hybrid (MaMTH) system to map the phospho-dependent ALK interactome. By screening a library of 86 SH2 domain-containing full length proteins, 30 novel ALK interactors were identified. Many of their interactions are correlated to ALK phosphorylation activity: oncogenic ALK mutations potentiate the interactions and ALK inhibitors attenuate the interactions. Among the novel interactors, NCK2 was further verified in neuroblastoma cells using co-immunoprecipitation. Modulation of ALK activity by addition of inhibitors lead to concomitant changes in the tyrosine phosphorylation status of NCK2 in neuroblastoma cells, strongly supporting the functionality of the ALK/NCK2 interaction. Our study provides a resource list of potential novel ALK signaling components for further study.

NPM1-ALK Overexpression-Driven Toxicity in ALCL Is Partner Dependent and Mediated By STAT1 Antagonism of STAT3 Survival Signaling, Revealing a Novel Therapeutic Strategy

Activation of the tyrosine-kinase domain (TKD) of anaplastic lymphoma kinase (ALK) drives malignant growth in subsets of several cancers as part of oncogenic fusions resulting from chromosomal rearrangements. Clinically, 70% of anaplastic large-cell lymphoma (ALCL) and ~5% of non-small-cell lung cancer (NSCLC) are ALK+. ALK tyrosine-kinase inhibitors (TKIs) are approved for treatment of NSCLC and are under investigation in ALCL. Clinical resistance in lung cancer typically develops within 10-12 months and is due most commonly to activation of alternate pathways or second-site kinase-domain mutations. In contrast, in ALK + ALCL, we and others have shown that over-expression of the NPM1-ALK fusion kinase is the major mechanism of TKI resistance. While this permits cells to survive TKI treatment, it results in a toxic signaling overdose upon inhibitor withdrawal both in vitro and in vivo. Therefore, NPM1-ALK is both essential to the survival of ALK+ ALCL and able to induce apoptosis when activated above a certain threshold. We now report the mechanism underlying NPM1-ALK signaling overdose.We find that all generations of ALK TKIs drive NPM1-ALK overexpression as the most frequent resistance mechanism in ALCL cells, but increased ALK activity upon inhibitor withdrawal is universally toxic. ALK stimulates several oncogenic pathways, including JAK/STAT3, MEK/ERK, and PI3K/AKT, but we find none of these canonical downstream pathways is a key driver of overdose toxicity. Novel analysis of viability data using a normalized weighted area under the curve to quantify level of rescue showed that only ALK inhibitors can rescue from ALK overdose signaling. We also observed that NPM1-ALK over-expression has a significantly more detrimental effect on cell fitness than the lung cancer fusion EML4-ALK, suggesting that the toxicity seen upon overdose is fusion-partner dependent. For confirmation, we generated constitutively active mutants of the ALK TKD with no fusion partner that are potently transforming but do not impair cell fitness upon ALK overdose. Phosphoproteomics analysis of resistant sub-clones during overdose as compared to parental lines in vehicle show an overlap of over 1,700 targets driven by ALK signaling. Furthermore, comparison of the parental line in drug to the resistant line undergoing overdose, 178 genes group together in the overall death state. These analyses revealed 21 phospho targets uniquely upregulated during ALK overdose (p <0.05). Annotation of these genes align with RNA-seq data showing increased phosphorylation of proteins driving the same pathways.STAT1 was a prominent target further pursued as the driver of apoptosis during NPMI-ALK overdose. Normally activated by INF-γ, STAT1 antagonizes the transcriptional output of STAT3, the core survival pathway of ALCL. It previously was shown to be a direct phospho target of NPM1-ALK, which at steady state results in its proteasomal degradation permitting predominance of the STAT3 transcriptional program. Upon NPM-ALK overdose, however, we see rapid and dramatic upregulation of phosphorylated STAT1 (Y705), in both the cytoplasm and nucleus. RNA-Seq analysis at the gene level shows loss of STAT3 targets but up-regulation of the STAT1 program, suggesting that during NPM1-ALK overdose STAT1 activation antagonizes STAT3 survival output required for ALCL cell viability and driving them to apoptosis. IFN-γ, already known to be detrimental to ALK+ ALCL cells as a single agent, combined with ALK inhibitors therefore represents a novel treatment strategy able to synergistically deactivate the core survival pathway of ALK+ ALCL. DisclosuresNo relevant conflicts of interest to declare.

Activated ALK Cooperates with N-Myc via Wnt/β-Catenin Signaling to Induce Neuroendocrine Prostate Cancer.

Neuroendocrine prostate cancer (NEPC) is an aggressive subtype of prostate cancer with poor prognosis, and there is a critical need for novel therapeutic approaches. NEPC is associated with molecular perturbation of several pathways, including amplification of MYCN. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase involved in the pathogenesis of neuroblastoma and other malignancies where it cooperates with N-Myc. We previously identified the first case of ALK F1174C-activating mutation in a patient with de novo NEPC who responded to the ALK inhibitor, alectinib. Here, we show that coactivation of ALK and N-Myc (ALK F1174C/N-Myc) is sufficient to transform mouse prostate basal stem cells into aggressive prostate cancer with neuroendocrine differentiation in a tissue recombination model. A novel gene signature from the ALK F1174C/N-Myc tumors was associated with poor outcome in multiple human prostate cancer datasets. ALK F1174C and ALK F1174C/N-Myc tumors displayed activation of the Wnt/β-catenin signaling pathway. Chemical and genetic ALK inhibition suppressed Wnt/β-catenin signaling and tumor growth in vitro in NEPC and neuroblastoma cells. ALK inhibition cooperated with Wnt inhibition to suppress NEPC and neuroblastoma proliferation in vitro and tumor growth and metastasis in vivo. These findings point to a role for ALK signaling in NEPC and the potential of cotargeting the ALK and Wnt/β-catenin pathways in ALK-driven tumors. Activated ALK and N-Myc are well known drivers in neuroblastoma development, suggesting potential similarities and opportunities to elucidate mechanisms and therapeutic targets in NEPC and vice versa. SIGNIFICANCE: These findings demonstrate that coactivation of ALK and N-Myc induces NEPC by stimulating the Wnt/β-catenin pathway, which can be targeted therapeutically.

Anaplastic lymphoma kinase inhibitor NVP‑TAE684 suppresses the proliferation of human pancreatic adenocarcinoma cells.

Anaplastic lymphoma kinase (ALK) is known to be an important therapeutic target in various types of cancer. NVP‑TAE684, a well‑known inhibitor of ALK, was revealed to exert antitumor effects in several different malignancies. However, the molecular mechanisms responsible for these antitumor effects in cancer cells, including pancreatic adenocarcinoma cells, remain unknown. In the present study, NVP‑TAE684 was investigated for its antitumor effects towards pancreatic adenocarcinoma cells. MTT assay, western blot analysis, flow cytometry, caspase‑3/7 activity assay and Trypan blue exclusion assay were used and it was revealed that NVP‑TAE684 suppressed the proliferation of seven human pancreatic adenocarcinoma cell lines (AsPC‑1, Panc‑1, MIA PaCa‑2, Capan‑1, CFPAC‑1, Colo‑357 and BxPC‑3), and significantly increased G2/M arrest and apoptotic cell death. Furthermore, NVP‑TAE684 inhibited the phosphorylation of ALK at Y1604, as well as that of downstream mediators such as AKT(S473) and ERK1/2(Y202/T204). Notably, knocking down ALK with siRNAs also decreased proliferation and promoted G2/M arrest and apoptosis. Furthermore, inhibition of ALK with NVP‑TAE684 or siRNA synergistically enhanced gemcitabine‑induced cell death by inducing apoptosis. In conclusion, the findings of the present study indicated that NVP‑TAE684 exerted its antitumor effects by inducing G2/M arrest and apoptosis via the inhibition of the ALK signaling pathway, and suggests its potential use as an antitumor agent against pancreatic adenocarcinoma.